Ultra-sensitive detection systems

ABSTRACT

Disclosed are compositions and methods for sensitive detection of one or multiple analytes. In general, the methods involve the use of special label components, referred to as reporter signals, that can be associated with, incorporated into, or otherwise linked to the analytes. In some embodiments, the reporter signals can be altered such that the altered forms of different reporter signals can be distinguished from each other. In some embodiments, sets of reporter signals can be used where two or more of the reporter signals in a set have one or more common properties that allow the reporter signals having the common property to be distinguished and/or separated from other molecules lacking the common property. In other embodiments, sets of reporter signal/analyte conjugates can be used where two or more of the reporter signal/analyte conjugates in a set have one or more common properties that allow the reporter signal/analyte conjugates having the common property to be distinguished and/or separated form other molecules lacking the common property. Reporter signals can also be in conjunction with analytes (such as in mixtures of reporter signals and analytes), where no significant physical association between the reporter signals and analytes occurs; or alone, where no analyte is present.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.09/929,266, filed Aug. 13, 2001, which claims benefit of U.S.Provisional Application No. 60/224,939, filed Aug. 11, 2000, and U.S.Provisional Application No. 60/283,498, filed Apr. 12, 2001. applicationSer. No. 09/929,266, filed Aug. 13, 2001; Application No. 60/224,939,filed Aug. 11, 2000; and Application No. 60/283,498, filed Apr. 12,2001, are hereby incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

This invention is generally in the field of detection of analytes andbiomolecules, and more specifically in the field of multiplex detectionand analysis of analytes and biomolecules.

BACKGROUND OF THE INVENTION

Detection of molecules is an important operation in the biological andmedical sciences. Such detection often requires the use of specializedlabel molecules, amplification of a signal, or both, because manymolecules of interest are present in low quantities and do not, bythemselves, produce detectable signals. Many labels, labeling systems,and signal amplification techniques have been developed. For example,nucleic acid molecules and sequences have been amplified and/or detectedusing polymerase chain reaction (PCR), ligase chain reaction (LCR),self-sustained sequence replication (3SR), nucleic acid sequence basedamplification (NASBA), strand displacement amplification (SDA), andamplification with Qβ replicase (Birkenmeyer and Mushahwar, J.Virological Methods, 35:117-126 (1991); Landegren, Trends Genetics9:199-202 (1993)). Proteins have been detected using antibody-baseddetection systems such as sandwich assays (Mailini and Maysef, “Asandwich method for enzyme immunoassay. I. Application to rat and humanalpha-fetoprotein” J. Immunol. Methods 8:223-234 (1975)) andenzyme-linked immunosorbent assays (Engvall and Perlmann, “Enzyme-linkedimmunosorbent assay (ELISA). Quantitative assay of immunoglobulin”Immunochemistry 8:871-874 (1971)), and two-dimensional (2-D) gelelectrophoresis (Patton, Biotechniques 28: 944-957 (2000)). Althoughthese techniques are useful, most have significant drawbacks andlimitations. For example, radioactive labels are dangerous and difficultto handle, fluorescent labels have limited capacity for multiplexdetection because of limitations on distinguishable labels, andamplification methods can be subject to spurious signal amplification.There is a need for improved detection labels and detection techniquesthat can detect minute quantities of specific molecules and that can behighly multiplexed.

Analysis of protein expression and presence, such as proteome profilingor proteomics, requires sensitive detection of multiple proteins.Current methods in proteome profiling suggests that there is a shortageof tools necessary for such detection (Haynes and Yates, Proteomeprofiling-pitfalls and progress. Yeast 17(2):81-87 (2000)). While thetechniques of chromatography and capillary electrophoresis are amenableto proteomic studies and have seen significant development efforts (seefor example, Krull et al., Specific applications of capillaryelectrochromatography to biopolymers, including proteins, nucleic acids,peptide mapping, antibodies, and so forth. J Chromatogr A, 887:137-63(2000), Hage, Affinity chromatography: a review of clinicalapplications. Clin Chem, 45(5):593-615 (1999), Hage et al.,Chromatographic Immunoassays., Anal Chem, 73(07):198 A-205 A, (2001),Krull et al., Labeling reactions applicable to chromatography andelectrophoresis of minute amounts of proteins. J Chromatogr B Biomed SciAppl, 699:173-208 (1997)), the workhorse of the industry remains twodimensional electrophoresis where the two dimensions are isoelectricfocusing and molecular size. Haynes and Yates point out the significantshortcomings of the technique but discuss the utility of the method inlight of such shortcomings. Hayes and Yates also discuss the techniquesof Isotope Coded Affinity Tags (ICAT), LC-LC-MS/MS, and stable isotopelabeling techniques (Shevchenko et al., Rapid ‘de novo’peptidesequencing by a combination of nanoelectrospray, isotopic labeling and aquadrupole/time-of-flight mass spectrometer. Rapid Commun Mass Spectrom11 (9):1015-1024 (1997); Oda et al., Accurate quantitation of proteinexpression and site-specific phosphorylation. Proc Natl Acad Sci USA96(12):6591-6596 (1999)).

Aebersold et al. (WO 00/11208) have described labels of the compositionPRG-L-A, where PRG is a protein reactive group, L is a linker (that maycontain isotopically distinguishable composition), and A is an affinitymoiety. Aebersold et al. describes a method where the protein reactivegroup is used to attach the label to a protein, an affinity capturemolecule is used to capture the affinity moiety, the remaining proteinsare discarded, then the affinity moiety is released and the labeledproteins are detected by mass spectrometry. The method of Aebersold etal. does not involve fragmentation or other modification of the labelsor proteins.

The technique of ICAT, where cysteine residues are labeled with heavy orlight tags that each contain affinity moieties, in control and testersamples, has received significant interest and holds potential forprotein profiling (Gygi et al., Quantitative analysis of complex proteinmixtures using isotope-coded affinity tags. Nat. Biotechnol.17(10):994-999 (1999), Griffin et al., Quantitative proteomic analysisusing a MALDI quadrupole time-of-flight mass spectrometer., Anal. Chem.,73:978-986 (2001)). Gygi et al. and Griffin et al. have demonstratedrelative profiling of two protein samples, where the two samples aredistinguished utilizing linkers containing either eight normal hydrogenor eight heavy hydrogen (deuterium) atoms. The relative concentrationsof labeled proteins are determined by ratio of peaks that are separatedby the corresponding 8 amu difference in the linker molecules. Currentimplementations have been limited to two labels. This technique does notinvolve fragmentation or other modification of the labels or proteins.

Mass spectrometry has been used to detect phosphorylated proteins(DeGnore and Qin, Fragmentation of phosphopeptides in an ion trap massspectrometer. J. Am. Soc. Mass Spectrom. 9:1175-1188 (1998); Qin andChait, Identification and characterization of posttranslationalmodifications of proteins by MALDI ion trap mass spectrometry. AnalChem, 69:4002-9 (1997); Annan et al., A multidimensional electrosprayMS-based approach to phosphopeptide mapping. Anal. Chem. 73:393-404(2001)). The methods make use of a signature mass to indicate thepresence of a phosphate group, for example m/z=63 and/or m/z=79corresponding to PO₂ ⁻ and PO₃ ⁻ ions in negative ion mode, or theneutral loss of 98 Daltons from the parent ion indicates the loss ofH₃PO₄ from the phosphorylated peptide, indicate phosphorylated Ser, Tyr,Thr. Once phosphorylated amino acids are identified, the peptidecontaining the modification is sequenced by standard MS/MS techniques.There is a need for a high reliability, highly multiplexed readoutsystem for proteomics.

The status of any living organism may be defined, at any given time inits lifetime, by the complex constellation of proteins that constituteits “proteome.” While the complete status of the proteome could bedefined by listing all proteins present (including modified variants) aswell as their intracellular locations and concentrations, such a task isbeyond the capabilities of any current single analytical method.

However, attempts have been made to define the status of a cell ortissue by identifying and measuring the relative concentrations of asmall subset of proteins. For example, Conrads et al., AnalyticalChemistry, 72:3349-3354 (2000), have described the use of “Accurate MassTags” (AMT) for proteome-wide protein identification. Conrads et al.show, for a simple organism, that a mass spectrometer of sufficient massaccuracy and resolution can be used to detect certain tryptic digestfragments from proteins. Once identified, the AMTs may be directlydetected in samples by tryptic digest of the proteins, and highaccuracy, high resolution mass spectrometry.

While the concept of Accurate Mass Tags is useful for protein discovery,as well as for generating peptide patterns in conventional biologicalexperiments, it does not solve the problem of sensitivity that is at theheart of a truly useful diagnostic multi-protein assessment. A usefulassessment consisting of AMTs will require samples containing a minimumof 2000 to 10,000 cells in order to permit reliable readout. This is sobecause many important cellular proteins are present at levels of only500 to 5000 molecules per cell. If a clinically relevant protein ispresent in 500 copies per cell, and a precious clinical sample from acancer patient contains only 1000 cells, the total number of proteins is500,000, an amount that lies below the limit of detection byconventional mass spectrometry. Thus, the types of measurements proposedby Conrads et al. for the study of proteomes after identification ofAMTs are not suitable for addressing important clinical problems such asthe diagnosis of cancer.

BRIEF SUMMARY OF THE INVENTION

Disclosed are compositions and methods for sensitive detection of one ormultiple analytes. In general, the methods involve the use of speciallabel components, referred to as reporter signals, that can beassociated with, incorporated into, or otherwise linked to the analytes.Reporter signals can also be used merely in conjunction with analytes,with no significant association between the analytes and reportersignals. Compositions where reporter signals are associated with,incorporated into, or otherwise linked to the analytes are referred toas reporter signal/analyte conjugates. Such conjugates include reportersignals associated with analytes, such as a reporter signal probehybridized to a nucleic acid sequence; reporter signals covalentlycoupled to analytes, such as reporter signals linked to proteins via alinking group; and reporter signals incorporated into analytes, such asfusions between a protein of interest and a peptide reporter signal.

In some embodiments, the reporter signals can be altered such that thealtered forms of different reporter signals can be distinguished fromeach other. Reporter signal/analyte conjugates can be altered, generallythrough alteration of the reporter signal portion of the conjugate, suchthat the altered forms of different reporter signals, altered forms ofdifferent reporter signal/analyte conjugates, or both, can bedistinguished from each other. Where the reporter signal or reportersignal/analyte conjugate is altered by fragmentation, any, some, or allof the fragments can be distinguished from each other, depending on theembodiment. For example, where reporter signals fragmented into twoparts, either or both parts of the reporter signals can bedistinguished. Where reporter signal/analyte conjugates are fragmentedinto two parts (with the break point in the reporter signal portion),either the reporter signal fragment, the reporter signal/analytefragment, or both can be distinguished. In some embodiments, only onepart of a fragmented reporter signal will be detected and so only thispart of the reported signals need be distinguished.

In some embodiments, sets of reporter signals can be used where two ormore of the reporter signals in a set have one or more common propertiesthat allow the reporter signals having the common property to bedistinguished and/or separated from other molecules lacking the commonproperty. In other embodiments, sets of reporter signal/analyteconjugates can be used where two or more of the reporter signal/analyteconjugates in a set have one or more common properties that allow thereporter signal/analyte conjugates having the common property to bedistinguished and/or separated form other molecules lacking the commonproperty. In still other embodiments, analytes can be fragmented (priorto or following conjugation) to produce reporter signal/analyte fragmentconjugates (which can be referred to as fragment conjugates). In suchcases, sets of fragment conjugates can be used where two or more of thefragment conjugates in a set have one or more common properties thatallow the fragment conjugates having the common property to bedistinguished and/or separated from other molecules lacking the commonproperty. It should be understood that fragmented analytes can beconsidered analytes in their own right. In this light, reference tofragmented analytes is made for convenience and clarity in describingcertain embodiments and to allow reference to both the base analyte andthe fragmented analyte.

As indicated above, reporter signals conjugated with analytes can bealtered while in the conjugate and distinguished. Conjugated reportersignals can also be dissociated or separated, in whole or in part, fromthe conjugated analytes prior to their alteration. Where the reportersignals are dissociated (in whole or in part) from the analytes, themethod can be performed such that the fact of association between theanalyte and reporter signal is part of the information obtained when thereporter signal is detected. In other words, the fact that the reportersignal may be dissociated from the analyte for detection does notobscure the information that the detected reporter signal was associatedwith the analyte.

Reporter signals can also be in conjunction with analytes (such as inmixtures of reporter signals and analytes), where no significantphysical association between the reporter signals and analytes occurs;or alone, where no analyte is present. In such cases, where reportersignals are not or are no longer associated with analytes, sets ofreporter signals can be used where two or more of the reporter signalsin a set have one or more common properties that allow the reportersignals having the common property to be distinguished and/or separatedfrom other molecules lacking the common property.

Detection of the reporter signals indicates the presence of thecorresponding analytes. The reporter signals preferably can have two keyfeatures. First, the reporter signals can be used in sets where all thereporter signals in the set have similar properties (for example, massspectrometry reporter signals may have similar mass-to-charge ratios).The similar properties allow the reporter signals to be distinguishedand/or separated from other molecules lacking one or more of theproperties. Second, all the reporter signals in a set can be fragmented,decomposed, reacted, derivatized, or otherwise modified to distinguishthe different reporter signals in the set. Preferably, mass spectrometryreporter signals are fragmented to yield fragments of similar charge butdifferent mass.

Differential distribution of mass in the fragments of the reportersignals can be accomplished in a number of ways. For example, reportersignals of the same nominal structure (for example, peptides having thesame amino acid sequence) can be made with different distributions ofheavy isotopes, such as deuterium; reporter signals of the same nominalstructure can be made with different distributions of modifications,such as methylation, phosphorylation, sulphation, and use ofseleno-methionine for methionine; reporter signals of the same nominalcomposition (for example, made up of the same amino acids) can be madewith different ordering of the subunits or components of the reportersignal; and reporter signals having the same nominal composition can bemade with a labile or scissile bond at a different location in thereporter signal. Each of these modes can be combined with each otherand/or one or more of the other modes to produce differentialdistribution of mass in the fragments of the reporter signals.

The reporter signals are preferably detected using mass spectrometrywhich allows sensitive distinctions between molecules based on theirmass-to-charge ratios. The disclosed reporter signals can be used asgeneral labels in myriad labeling and/or detection techniques. A set ofisobaric reporter signals can be used for multiplex labeling and/ordetection of many analytes since the reporter signal fragments can bedesigned to have a large range of masses, with each mass individuallydistinguishable upon detection.

The disclosed method has advantageous properties which can be used as adetection system in a number of fields, including antibody or proteinmicroarrays, DNA microarrays, expression profiling, comparativegenomics, immunology, diagnostic assays, and quality control.

A. Reporter Molecule Labeling

In one form of the disclosed method, referred to as reporter moleculelabeling (RML), reporter signals are first associated with the analytesand then dissociated and detected. The reporter signals preferably areassociated with the analytes via interaction of specific bindingmolecules with the analytes. The reporter signals are either directly orindirectly associated with the specific binding molecules such thatinteraction of the specific binding molecules with the analytes allowsthe reporter signals to be associated with the analytes. The method canbe performed such that the fact of association between the analyte andreporter signal is part of the information obtained when the reportersignal is detected. In other words, the fact that the reporter signalmay be dissociated from the analyte for detection does not obscure theinformation that the detected reporter signal was associated with theanalyte.

B. Reporter Signal Labeling

In another form of the disclosed method, referred to as reporter signallabeling (RSL), reporter signals are used for sensitive detection of oneor multiple analytes. In the method, analytes labeled with reportersignals are analyzed using the reporter signals to distinguish thelabeled analytes (where the analytes are labeled with the reportersignals). Detection of the reporter signals indicates the presence ofthe corresponding analyte(s). The detected analyte(s) can then beanalyzed using known techniques. The labels provide a uniqueanalyte/label composition that can specifically identify the analyte(s).This is accomplished through the use of the specialized reporter signalsas the labels. The labeled analyte(s) can be fragmented prior toanalysis. An analyte sample to be analyzed can also be subjected tofractionation or separation to reduce the complexity of the samples.Fragmentation and fractionation can also be used together in the sameassay. Such fragmentation and fractionation can simplify and extend theanalysis of the analytes.

Reporter signals can be coupled or directly associated with an analyte.For example, a reporter signal can be coupled to an analyte via reactivegroups, or a reporter molecule (composed of a specific binding moleculeand a reporter signal) can be associated with an analyte. The reportersignals can be attached to analytes in any manner. For example, reportersignals can be covalently coupled to proteins through a sulfur-sulfurbond between a cysteine on the protein and a cysteine on the reportersignal. Many other chemistries and techniques for coupling compounds toanalytes are known and can be used to couple reporter signals toanalytes. For example, coupling can be made using thiols, epoxides,nitriles for thiols, NHS esters, isothiocyanates for amines, andalcohols for carboxylic acids. Reporter signals can be attached toanalytes either directly or indirectly, for example, via a linker.

Alternatively, a reporter signal can be associated with an analyteindirectly. In this mode, a “coding” molecule containing a specificbinding molecule and a coding tag can be associated with the analyte(via the specific binding molecule). Alternatively, a coding tag can becoupled or directly associated with the analyte. Then a reporter signalassociated with a decoding tag (such a combination is another form ofreporter molecule) is associated with the coding molecule through aninteraction between the coding tag and the decoding tag. An example ofthis interaction is hybridization where the coding and decoding tags arecomplementary nucleic acid sequences. The result is an indirectassociation of the reporter signal with the analyte. This mode has theadvantage that all of the interactions of the reporter signals with thecoding molecule can be made chemically and physically similar by usingthe same types of coding tags and decoding tags for all of the codingmolecules and reporter molecules in a set.

Reporter signals can be fragmented, decomposed, reacted, derivatized, orotherwise modified, preferably in a characteristic way. This allows ananalyte to which the reporter signal is attached to be identified by thecorrelated detection of the labeled analyte and one or more of theproducts of the labeled analyte following fragmentation, decomposition,reaction, derivatization, or other modification of the reporter signal(the labeled analyte is the analyte/reporter signal combination). Thealteration of the reporter signal will alter the labeled analyte in acharacteristic and detectable way. Together, the detection of acharacteristic labeled analyte and a characteristic product of thelabeled analyte can uniquely identify the analyte. In this way, usingthe disclosed method and materials, one or more analytes can bedetected, either alone or together (for example, in a multiplex assay).Further, one or more analytes in one or more samples can be detected ina multiplex manner. Preferably, for mass spectrometry reporter signals,the reporter signals are fragmented to yield fragments of similar chargebut different mass.

In preferred embodiments, reporter signals are used in sets where allthe reporter signals in the set have similar properties (such as similarmass-to-charge ratios). The similar properties allow the reportersignals to be distinguished and/or separated from other moleculeslacking one or more of the properties. Alternatively, or in addition,reporter signals can be used in sets such that the resulting labeledanalytes will have similar properties allowing the labeled analytes tobe distinguished and/or separated from other molecules lacking one ormore of the properties.

Analytes can be detected using the disclosed reporter signals in avariety of ways. For example, the analyte and attached reporter signalcan be detected together, one or more fragments of the analyte and theattached reporter signal(s) can be detected together, the fragments ofthe reporter signal can be detected, or a combination. Preferreddetection involves detection of the analyte/reporter signal both beforeand after fragmentation of the reporter signal.

A preferred form of the disclosed method involves correlated detectionof the reporter signals both before and after fragmentation of thereporter signal. This allows labeled analytes to be detected andidentified via the change in labeled analyte. That is, the nature of thereporter signal detected (non-fragmented versus fragmented) identifiesthe analyte as labeled. Where the analytes and reporter signals aredetected by mass-to-charge ratio, the change in mass-to-charge ratiobetween fragmented and non-fragmented samples provides the basis forcomparison. Such mass-to-charge ratio detection is preferablyaccomplished with mass spectrometry.

As an example, an analyte in a sample can be labeled with reportersignal designed as a mass spectrometry label. The labeled analyte can besubjected to mass spectrometry. A peak corresponding to theanalyte/reporter signal will be detected. Fragmentation of the reportersignal in a collision cell in the mass spectrometer would result in ashift in the peak corresponding to the loss of a portion of the attachedreporter signal, the appearance of a peak corresponding to the lostfragment, or a combination of both events. Significantly, the shiftobserved will depend on which reporter signal is on the analyte sincedifferent reporter signals will, by design, produce fragments withdifferent mass-to-charge ratios. The combination event of detection ofthe parent mass-to-charge (with no collision gas) and the mass-to-chargecorresponding to the loss of the fragment from the reporter signal (withcollision gas) indicates a labeled analyte. The identity of the analytecan be determined by standard mass spectrometry techniques, such ascompositional analysis.

A powerful form of the disclosed method is use of analytes labeled withreporter signals to assay multiple samples (for example, time seriesassays or other comparative analyses). Knowledge of the temporalresponse of a biological system following perturbation is a verypowerful process in the pursuit of understanding the system. To followthe temporal response, a sample of the system is obtained (for example,cells from a cell culture, mice initially synchronized and sacrificed)at determined times following the perturbation. Knowledge of spatialanalyte profiles (for example, relative position within a tissuesection) is a very powerful process in the pursuit of understanding thebiological system.

In the disclosed method a series of samples can each be labeled with adifferent reporter signal from a set of reporter signals. Preferredreporter signals for this purpose would be those using differentiallydistributed mass. In particular, the use of stable isotopes is preferredto ensure that members of the set of reporter signals would behavechemically identically and yet would be distinguishable.

The labeled analytes are preferably detected using mass spectrometrywhich allows sensitive distinctions between molecules based on theirmass-to-charge ratios. The disclosed reporter signals can be used asgeneral labels in myriad labeling and/or detection techniques. A set ofisobaric reporter signals can be used for multiplex labeling and/ordetection of many analytes since the reporter signal fragments can bedesigned to have a large range of masses, with each mass individuallydistinguishable upon detection. Where the same analyte or type ofanalyte is labeled with a set of isobaric reporter signals (by, forexample, labeling the same analyte in different samples), the set oflabeled analytes that results from use of an isobaric set of reportersignals will also be isobaric. Fragmentation of the reporter signalswill split the set of labeled analytes into individually detectablelabeled proteins of characteristically different mass.

The disclosed method can be used in many modes. For example, thedisclosed method can be used to detect a specific analyte (in a specificsample or in multiple samples) or multiple analytes (in a single sampleor multiple samples). In each case, the analyte(s) to be detected can beseparated either from other, unlabeled analytes or from other moleculeslacking a property of the labeled analyte(s) to be detected. Forexample, analytes in a sample can be generally labeled with reportersignals and some analytes can be separated on the basis of some propertyof the analytes. For example, the separated analytes could have acertain mass-to-charge ratio (separation based on mass-to-charge ratiowill select both labeled and unlabeled analytes having the selectedmass-to-charge ratio). As another example, all of the labeled analytescan be distinguished and/or separated from unlabeled molecules based ona feature of the reporter signal such as an affinity tag. Wheredifferent affinity tags are used, some labeled analytes can bedistinguished and/or separated from others. Reporter signal labelingallows profiling of analytes and cataloging of analytes.

In one mode of the disclosed method, multiple analytes in multiplesamples are labeled where all of the analytes in a given sample arelabeled with the same reporter signal. That is, the reporter signal isused as a general label of the analytes in a sample. Each sample,however, uses a different reporter signal. This allows samples as awhole to be compared with each other. By additionally separating ordistinguishing different analytes in the samples, one can easily analyzemany analytes in many samples in a single assay. For example, proteinsin multiple samples can be labeled with reporter signals as describedabove, and the samples mixed together. If some or all of the variouslabeled proteins are separated by, for example, association of theproteins with antibodies on an array, the presence and amount of a givenprotein in each of the samples can be determined by identifying thereporter signals present at each array element. If the proteincorresponding to a given array element was present in a particularsample, then some of the protein associated with that array element willbe labeled with the reporter signal used to label that particularsample. Detection of that reporter signal will indicate this. This samerelationship holds true for all of the other samples. Further, theamount of reporter signal detected can indicate the amount of a givenprotein in a given sample, and the simultaneous quantitation of proteinin multiple samples can provide a particularly accurate comparison ofthe levels of the proteins in the various samples.

In one form of reporter signal labeling, referred to as reporter signalprotein labeling (RSPL), reporter signals are used for sensitivedetection of one or multiple proteins. In the method, proteins labeledwith reporter signals are analyzed using the reporter signals todistinguish the labeled proteins. Detection of the reporter signalsindicates the presence of the corresponding protein(s). The detectedprotein(s) can then be analyzed using known techniques. The labelsprovide a unique protein/label composition that can specificallyidentify the protein(s). This is accomplished through the use of thespecialized reporter signals as the labels. The labeled protein(s) canbe fragmented, such as by protease digestion, prior to analysis. Aprotein sample to be analyzed can also be subjected to fractionation orseparation to reduce the complexity of the samples. Fragmentation andfractionation can also be used together in the same assay. Suchfragmentation and fractionation can simplify and extend the analysis ofthe proteins.

C. Reporter Signal Calibration

In another form of the method, referred to as reporter signalcalibration (RSC), a form of reporter signals referred to as reportersignal calibrators are mixed with analytes or analyte fragments, thereporter signal calibrators and the analytes or analyte fragments arealtered, and the altered forms of the reporter signal calibrators andaltered forms of the analytes or analyte fragments are detected.Reporter signal calibrators are useful as standards for assessing theamount of analytes present. That is, one can add a known amount of areporter signal calibrator in order to assess the amount of analytepresent comparing the amount of altered analyte or analyte fragmentdetected with the amount of altered reporter signal calibrator detectedand calibrating these amounts with the known amount of reporter signalcalibrator added (and thus the predicted amount of altered reportersignal calibrator).

In some embodiments, each analyte or analyte fragment can share one ormore common properties with at least one reporter signal calibrator suchthat the reporter signal calibrators and analytes or analyte fragmentshaving the common property can be distinguished and/or separated fromother molecules lacking the common property.

In some embodiments, reporter signal calibrators and analytes andanalyte fragments can be altered such that the altered form of ananalyte or analyte fragment can be distinguished from the altered formof the reporter signal calibrator with which the analyte or analytefragment shares a common property. In some embodiments, the alteredforms of different reporter signal calibrators can be distinguished fromeach other. In some embodiments, the altered forms of different analytesor analyte fragments can be distinguished from each other.

In some embodiments of reporter signal calibration, the analyte oranalyte fragment is not altered and so the altered reporter signalcalibrators and the analytes or analyte fragments are detected. In thiscase, the analyte or analyte fragment can be distinguished from thealtered form of the reporter signal calibrator with which the analyte oranalyte fragment shares a common property.

In some embodiments the analyte or analyte fragment may be a reportersignal or a fragment of a reporter signal. In this case, the reportersignal calibrators serve as calibrators for the amount of reportersignal detected.

Reporter signal calibration is preferably used in connection withproteins and peptides (as the analytes). This form of reporter signalcalibration is referred to as reporter signal protein calibration.Reporter signal protein calibration is useful, for example, forproducing protein signatures of protein samples. As used herein, aprotein signature is the presence, absence, amount, or presence andamount of a set of proteins or protein surrogates.

In some embodiments of reporter signal protein calibration, the presenceof labile, scissile, or cleavable bonds in the proteins to be detectedcan be exploited. Peptides, proteins, or protein fragments (collectivelyreferred to, for convenience, as protein fragments in the remainingdescription) containing such bonds can be altered by fragmentation atthe bond. In this way, reporter signal calibrators having a commonproperty (such as mass-to-charge ratio) with the protein fragments canbe used and the altered forms of the reporter signal calibrators and thealtered (that is, fragmented) forms of the protein fragments can bedetected and distinguished. In this regard, although the proteinfragments share a common property with their matching reporter signalcalibrators, the altered forms of the reporter signal calibrators andaltered forms of protein fragments can be distinguished (because, forexample, the altered forms have different properties, such as differentmass-to-charge ratios).

D. Reporter Signal Fusions

In another form of the disclosed method and compositions, referred to asreporter signal fusions (RSF), reporter signal peptides are joined witha protein or peptide of interest in a single amino acid segment. Suchfusions of proteins with reporter signal peptides can be expressed froma nucleic acid molecule encoding the amino acid segment that constitutesthe fusion. The fusion protein is referred to herein as a reportersignal fusion. The reporter signal peptides allow for sensitivemonitoring and detection of the proteins and peptides to which they arefused, and of expression of the genes, vectors, expression constructs,and nucleic acids that encode them. In particular, the reporter signalfusions allow sensitive and multiplex detection of expression ofparticular proteins and peptides of interest, and/or of the genes,vectors, and expression constructs encoding the proteins and peptides ofinterest.

The disclosed reporter signal fusions also are useful for creatingcells, cell lines, and organisms that have particular protein(s),gene(s), vector(s), and/or expression sequence(s) labeled (that is,associated with or involved in) reporter signal fusions. For example, aset of nucleic acid constructs, each encoding a reporter signal fusionwith a different reporter signal peptide, can be used to uniquely labela set of cells, cell lines, and/or organisms. Processing of a samplefrom any of the labeled sources can result in a unique altered form ofthe reporter signal peptide (or the amino acid segment or an amino acidsubsegment) for each of the possible sources that can be distinguishedfrom the other altered forms. Detection of a particular altered formidentifies the source from which it came.

The disclosed reporter signal fusions also can be used to “label”particular pathways, regulatory cascades, and other suites of genes,proteins, vectors, and/or expressions sequences. Such labeling can bewithin the same cell, cell line, or organism, or across a set of cells,cell lines, or organisms. For example, nucleic acid segments encodingreporter signal fusions can be associated with endogenous expressionsequences of interest, endogenous genes of interest, exogenousexpression sequences of interest, exogenous genes of interest, or acombination. The exogenous constructs then are introduced into the cellsor organisms of interest. The association with endogenous expressionsequences or genes can be accomplished, for example, by introducing anucleic acid molecule (encoding the reporter signal fusion) forinsertion at the site of the expression sequences or gene of interest,or by creating a vector or other nucleic acid construct (containing boththe endogenous expression sequences or gene and a nucleic acid segmentencoding the reporter signal fusion) in vitro and introducing theconstruct into the cells or organisms of interest. Many other uses andmodes of use are possible, a number of which are described in theillustrations below. In particular, the disclosed reporter signalfusions can be used in any context and for any purpose that greenfluorescent protein and green fluorescent protein fusions are used.However, the disclosed reporter signal proteins have more uses and aremore useful than green fluorescent protein at least because of theability to multiplex the disclosed reporter signal fusions.

The reporter signal peptides can be used for sensitive detection of oneor multiple proteins (that is, the proteins to which the reporter signalpeptides are fused). In the method, proteins fused with reporter signalpeptides are analyzed using the reporter signal peptides to distinguishthe reporter signal fusions. Detection of the reporter signal peptidesindicates the presence of the corresponding protein(s). The detectedprotein(s) can then be analyzed using known techniques. The reportersignal fusions provide a unique protein/label composition that canspecifically identify the protein(s). This is accomplished through theuse of the specialized reporter signal peptides as the labels.

The reporter signal fusions can be produced by expression from nucleicacid molecules encoding the fusions. Thus, the disclosed fusionsgenerally can be designed by designing nucleic acid segments that encodeamino acid segments where the amino acid segments comprise a reportersignal peptide and a protein or peptide of interest. A given nucleicacid molecule can comprise one or more nucleic acid segments. A givennucleic acid segment can encode one or more amino acid segments. A givenamino acid segment can include one or more reporter signal peptides andone or more proteins or peptides of interest. The disclosed amino acidsegments consist of a single, contiguous polypeptide chain. Thus,although multiple amino acid segments can be part of the same contiguouspolypeptide chain, all of the components (that is, the reporter signalpeptide(s) and protein(s) and peptide(s) of interest) of a given aminoacid segment are part of the same contiguous polypeptide chain.

Reporter signal peptides can be fragmented, decomposed, reacted,derivatized, or otherwise modified, preferably in a characteristic way.This allows a protein to which the reporter signal peptide is fused tobe identified by detection of one or more of the products of thereporter signal fusion following fragmentation, decomposition, reaction,derivatization, or other modification of the reporter signal peptide.Expression of one or more proteins in one or more samples can bedetected in a multiplex manner. Preferably, for mass spectrometryreporter signals, the reporter signal peptides are fragmented to yieldfragments of similar charge but different mass.

Preferably, the reporter signal peptides are fragmented to yieldfragments of similar charge but different mass. This allows eachreporter signal fusion (and/or each reporter signal peptide) in a set tobe distinguished by the different mass-to-charge ratios of the fragmentsof (that is, altered forms of) the reporter signal peptides or reportersignal fusions. This is possible since the fragments of the differentreporter signal peptides (or the fragments of the reporter signalfusions) can be designed to have different mass-to-charge ratios. In thedisclosed method, this allows each reporter signal fusion to bedistinguished by the mass-to-charge ratios of the reporter signalfusions after fragmentation of the reporter signal peptide.

Alteration of reporter signals peptides in reporter signal fusions canproduce a variety of altered compositions. Any or all of these alteredforms can be detected. For example, the altered form of the reportersignal peptide can be detected, the altered form of the amino acidsegment (which contains the reporter signal peptide) can be detected,the altered form of a subsegment of the amino acid segment can bedetected, or a combination of these can be detected. Where the reportersignal peptide is altered by fragmentation, the result generally will bea fragment of the reporter signal peptide and an altered form of theamino acid segment containing the protein or peptide of interest and aportion of the reporter signal peptide (that is, the portion not in thereporter signal peptide fragment). The protein or peptide of interestalso can be fragmented. The result would be a subsegment of the aminoacid segment. The amino acid subsegment would contain the reportersignal peptide and a portion of the protein or peptide of interest. Whenthe reporter signal peptide in an amino acid subsegment is altered(which can occur before, during, or after fragmentation of the aminoacid segment), the result is an altered form of the amino acidsubsegment (and an altered form of the reporter signal peptide). Thisaltered form of amino acid subsegment can be detected.

As with reporter signals generally, reporter signal peptides can be usedin sets where the reporter signal peptides in a set can have one or morecommon properties that allow the reporter signal peptides to beseparated or distinguished from molecules lacking the common property.In the case of reporter signal fusions, amino acid segments and aminoacid subsegments can be used in sets where the amino acid segments andamino acid subsegments in a set can have one or more common propertiesthat allow the amino acid segments and amino acid subsegments,respectively, to be separated or distinguished from molecules lackingthe common property. In general, the component(s) of the reporter signalfusions having common properties can depend on the component(s) to bedetected and/or the mode of the method being used.

Nucleic acid molecules encoding reporter signal fusions can be used insets where the reporter signal peptides in the reporter signal fusionsencoded by a set of nucleic acid molecules can have one or more commonproperties that allow the reporter signal peptides to be separated ordistinguished from molecules lacking the common property. Similarly,nucleic acid segments (which, generally, are part of nucleic acidmolecules) encoding reporter signal fusions can be used in sets wherethe reporter signal peptides in the reporter signal fusions encoded by aset of nucleic acid segments can have one or more common properties thatallow the reporter signal peptides to be separated or distinguished frommolecules lacking the common property. Other relationships betweenmembers of the sets of nucleic acid molecules, nucleic acid segments,amino acid segments, reporter signal peptides, and proteins of interestare contemplated.

Cells, cell lines, organisms, and expression of genes and proteins canbe detected using the disclosed reporter signal fusions in a variety ofways. For example, the protein and attached reporter signal peptide canbe detected together, one or more peptides of the protein and theattached reporter signal peptide(s) can be detected together, thefragments of the reporter signal peptide can be detected, or acombination. Preferred detection involves detection of the reportersignal fusion both before and after fragmentation of the reporter signalpeptide.

A powerful form of the disclosed method is use of reporter signalfusions to assay multiple samples (for example, time series assays orother comparative analyses). Knowledge of the temporal response of abiological system following perturbation is a very powerful process inthe pursuit of understanding the system. To follow the temporal responsea sample of the system is obtained (for example, cells from a cellculture, mice initially synchronized and sacrificed) at determined timesfollowing the perturbation. Knowledge of spatial protein profiles (forexample, relative position within a tissue section) is a very powerfulprocess in the pursuit of understanding the biological system.

The reporter signal fusions are preferably detected using massspectrometry which allows sensitive distinctions between molecules basedon their mass-to-charge ratios. A set of isobaric reporter signalpeptides or reporter signal fusions can be used for multiplex labelingand/or detection of the expression of many genes, proteins, vectors,expression constructs, cells, cell lines, and organisms since thereporter signal peptide fragments can be designed to have a large rangeof masses, with each mass individually distinguishable upon detection.Where the same gene, protein, vector, expression construct, cell, cellline, or organism (or the same type of gene, protein, vector, expressionconstruct, cell, cell line, or organism) is labeled with a set ofreporter signal fusions that are isobaric or that include isobaricreporter signal peptides (by, for example, “labeling” the same gene,protein, vector, expression construct, cell, cell line, or organism indifferent samples), the set of reporter signal fusions or reportersignal peptides that results will also be isobaric. Fragmentation of thereporter signal peptides will split the set of reporter signal peptidesinto individually detectable reporter signal fusion fragments andreporter signal peptide fragments of characteristically different mass.

A preferred form of the disclosed method involves filtering of isobaricreporter signal fusions or reporter signal peptides from other moleculesbased on mass-to-charge ratio, fragmentation of the reporter signalpeptides to produce fragments having different masses, and detection ofthe different fragments based on their mass-to-charge ratios. The methodis best carried out using a tandem mass spectrometer as describedelsewhere herein.

It is an object of the present invention to provide a method for themultiplexed determination of presence, amount, or presence and amount ofanalytes.

It is an object of the present invention to provide labeled proteinssuch that the presence, amount, or presence and amount of the proteinscan be determined.

It is another object of the present invention to provide a method forlabeling proteins so as to allow the multiplexed determination ofpresence, amount, or presence and amount of proteins.

It is another object of the present invention to provide a method forthe multiplexed determination of presence, amount, or presence andamount of proteins.

It is an object of the present invention to provide a method fordetecting a mass tag signature.

It is an object of the present invention to provide a method fordetecting a protein signature.

It is another object of the present invention to provide an assessmentof the identity and purity of the peptides comprising a proteinsignature.

It is another object of the present invention to provide a method fordetecting phosphopeptides, or other posttranslational proteinmodifications, among the peptides comprising a protein signature.

It is another object of the present invention to provide kits forgenerating mass tag signatures.

It is another object of the present invention to provide kits forgenerating protein signatures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are graphs of mass-to-charge ratio (m/z) versus signalintensity. FIG. 1A shows the results where there is no fragmentation ofthe reporter signal. A single peak represents the parent ion. FIG. 1Bshows the results where the reporter signal is fragmented. The parention along with two fragmentation ions are detected.

FIGS. 2A and 2B are graphs of mass-to-charge ratio (m/z) versus detectedcounts. FIG. 2A shows the results where no fragmentation of reportersignals A and B occurs. FIG. 2B shows the results where all of thereporter signals are fragmented (A fragments to A1 and A2, B fragmentsto B1 and B2).

FIG. 3 is an example of an ESI-TOF mass spectrum of an example of areporter signal peptide (LAT3838 in this case). Most of the complexityof the spectrum comes from fragmentation of the reporter signal peptidein the source.

FIG. 4 is an example a spectrum of a selected reporter signal peptide(LAT3838 in this case) following fragmentation. The parent reportersignal was selected at a filter setting of m/z=1044 and altered byfragmented by collision with argon gas at about 20 eV collision energy.The daughter reporter signal peptide fragment at m/z=600 corresponds tothe expected PAGSLR⁺ fragment (amino acids 6-11 of SEQ ID NO:2).

FIG. 5 is an example of a spectrum of the fragmentation products of fivereporter signal peptides (LAT3838 and LAT3843 through LAT3856). Thepeaks corresponding to the reporter signal peptide fragments of each arelabeled.

FIG. 6 is an example of a spectrum showing the effect of the loss of aphosphate group from reporter signal peptide fragments.

FIG. 7 is an example of a spectrum showing differentiation of reportersignal peptide fragments based on the use of stable isotopes in thereporter signal peptides.

FIG. 8 is an example of a spectrum of a complex mixture of moleculesthat includes five reporter signal peptides (m/z=1389.6).

FIGS. 9A and 9B are an example of a spectrum of a set of reporter signalpeptides following selection based on mass-to-charge ratio (m/z around1390) and illustrating a nearly non-existent background everywhereexcept at m/z around 1390. This is the same sample (prior to selection)from which the complex spectrum of FIG. 8 was obtained. FIG. 9B is anexpanded view of the peak. Selection was not perfect in this example asthe finite resolution of the filter allowed three peaks to pass.

FIG. 10 is an example of a spectrum of the fragmentation products of theselected reporter signal peptides of FIG. 9 (originally in the complexsample of FIG. 8). Five prominent peaks of approximately the samemagnitude appear at the expected m/z of the reporter signal peptidefragments of each of the five reporter signal peptides. Unfragmentedreporter signal peptides remain in the rightmost peak (m/z near 1390).

DETAILED DESCRIPTION OF THE INVENTION

Disclosed are compositions and methods for sensitive detection of one ormultiple analytes. In general, the methods involve the use of speciallabel components, referred to as reporter signals, that can beassociated with, incorporated into, or otherwise linked to the analytes.Reporter signals can also be used merely in conjunction with analytes,with no significant association between the analytes and reportersignals. Compositions where reporter signals are associated with,incorporated into, or otherwise linked to the analytes are referred toas reporter signal/analyte conjugates. Such conjugates include reportersignals associated with analytes, such as a reporter signal probehybridized to a nucleic acid sequence; reporter signals covalentlycoupled to analytes, such as reporter signals linked to proteins via alinking group; and reporter signals incorporated into analytes, such asfusions between a protein of interest and a peptide reporter signal.

In some embodiments, the reporter signals can be altered such that thealtered forms of different reporter signals can be distinguished fromeach other. Reporter signal/analyte conjugates can be altered, generallythrough alteration of the reporter signal portion of the conjugate, suchthat the altered forms of different reporter signals, altered forms ofdifferent reporter signal/analyte conjugates, or both, can bedistinguished from each other. Where the reporter signal or reportersignal/analyte conjugate is altered by fragmentation, any, some, or allof the fragments can be distinguished from each other, depending on theembodiment. For example, where reporter signals fragmented into twoparts, either or both parts of the reporter signals can bedistinguished. Where reporter signal/analyte conjugates are fragmentedinto two parts (with the break point in the reporter signal portion),either the reporter signal fragment, the reporter signal/analytefragment, or both can be distinguished. In some embodiments, only onepart of a fragmented reporter signal will be detected and so only thispart of the reported signals need be distinguished.

In some embodiments, sets of reporter signals can be used where two ormore of the reporter signals in a set have one or more common propertiesthat allow the reporter signals having the common property to bedistinguished and/or separated from other molecules lacking the commonproperty. In other embodiments, sets of reporter signal/analyteconjugates can be used where two or more of the reporter signal/analyteconjugates in a set have one or more common properties that allow thereporter signal/analyte conjugates having the common property to bedistinguished and/or separated form other molecules lacking the commonproperty. In still other embodiments, analytes can be fragmented (priorto or following conjugation) to produce reporter signal/analyte fragmentconjugates (which can be referred to as fragment conjugates). In suchcases, sets of fragment conjugates can be used where two or more of thefragment conjugates in a set have one or more common properties thatallow the fragment conjugates having the common property to bedistinguished and/or separated form other molecules lacking the commonproperty. It should be understood that fragmented analytes can beconsidered analytes in their own right. In this light, reference tofragmented analytes is made for convenience and clarity in describingcertain embodiments and to allow reference to both the base analyte andthe fragmented analyte.

As indicated above, reporter signals conjugated with analytes can bealtered while in the conjugate and distinguished. Conjugated reportersignals can also be dissociated or separated, in whole or in part, fromthe conjugated analytes prior to their alteration. Where the reportersignals are dissociated (in whole or in part) from the analytes, themethod can be performed such that the fact of association between theanalyte and reporter signal is part of the information obtained when thereporter signal is detected. In other words, the fact that the reportersignal may be dissociated from the analyte for detection does notobscure the information that the detected reporter signal was associatedwith the analyte.

Reporter signals can also be in conjunction with analytes (such as inmixtures of reporter signals and analytes), where no significantphysical association between the reporter signals and analytes occurs;or alone, where no analyte is present. In such cases, where reportersignals are not or are no longer associated with analytes, sets ofreporter signals can be used where two or more of the reporter signalsin a set have one or more common properties that allow the reportersignals having the common property to be distinguished and/or separatedfrom other molecules lacking the common property.

Detection of the reporter signals indicates the presence of thecorresponding analytes. The reporter signals can have two key features.First, the reporter signals can be used in sets where all the reportersignals in the set have similar properties. The similar properties allowthe reporter signals to be distinguished and/or separated from othermolecules lacking one or more of the properties. Preferably, thereporter signals in a set have the same mass-to-charge ratio (m/z). Thatis, the reporter signals in a set are isobaric. This allows the reportersignals to be separated precisely from other molecules based onmass-to-charge ratio. The result of the filtering is a huge increase inthe signal to noise ratio (S/N) for the system, allowing more sensitiveand accurate detection.

Second, all the reporter signals in a set can be fragmented, decomposed,reacted, derivatized, or otherwise modified to distinguish the differentreporter signals in the set. For example, the reporter signals can befragmented to yield fragments having the same charge but different mass.This allows each reporter signal in a set to be distinguished by thedifferent mass-to-charge ratios of the fragments of the reportersignals. This is possible since, although the unfragmented reportersignals in a set are isobaric, the fragments of the different reportersignals are not. Reporter signals to be detected on the basis ofmass-to-charge ratio and/or to be detected with the use of a massspectrometer, can be referred to as mass spectrometer reporter signals.

Differential distribution of mass in the fragments of the reportersignals can be accomplished in a number of ways. For example, reportersignals of the same nominal structure (for example, peptides having thesame amino acid sequence), can be made with different distributions ofheavy isotopes, such as deuterium. All reporter signals in the set wouldhave the same number of a given heavy isotope, but the distribution ofthese would differ for different reporter signals. Similarly, reportersignals of the same general structure (for example, peptides having thesame amino acid sequence), can be made with different distributions ofmodifications, such as methylation, phosphorylation, sulphation, and useof seleno-methionine for methionine. All reporter signals in the setwould have the same number of a given modification, but the distributionof these would differ for different reporter signals. Reporter signalsof the same nominal composition (for example, made up of the same aminoacids), can be made with different ordering of the subunits orcomponents of the reporter signal. All reporter signals in the set wouldhave the same number of subunits or components, but the distribution ofthese would be different for different reporter signals. Reportersignals having the same nominal composition (for example, made up of thesame amino acids), can be made with a labile or scissile bond at adifferent location in the reporter signal. All reporter signals in theset would have the same number and order of subunits or components.Where the labile or scissile bond is present between particular subunitsor components, the order of subunits or components in the reportersignal can be the same except for the subunits or components creatingthe labile or scissile bond. Each of these modes can be combined withone or more of the other modes to produce differential distribution ofmass in the fragments of the reporter signals. For example, differentdistributions of heavy isotopes can be used in reporter signals where alabile or scissile bond is placed in different locations.

The reporter signals are preferably detected using mass spectrometrywhich allows sensitive distinctions between molecules based on theirmass-to-charge ratios. The disclosed reporter signals can be used asgeneral labels in myriad labeling and/or detection techniques. A set ofisobaric reporter signals can be used for multiplex labeling and/ordetection of many analytes since the reporter signal fragments can bedesigned to have a large range of masses, with each mass individuallydistinguishable upon detection.

Current technologies are limited in their ability to multiplex labels.In contrast, the disclosed method allows the readout of many samplessimultaneously and high internal accuracy in comparison to a sequentialreadout system.

A preferred form of the disclosed method involves filtering of isobaricreporter signals from other molecules based on mass-to-charge ratio,fragmentation of the reporter signals to produce fragments havingdifferent masses, and detection of the different fragments based ontheir mass-to-charge ratios. The method is best carried out using atandem mass spectrometer where the isobaric reporter signals are passedthrough a filtering quadrupole, the reporter signals are fragmented in acollision cell, and the fragments are distinguished and detected in atime-of-flight (TOF) stage. In such an instrument the sample is ionizedin the source (for example, in a MALDI ion source) to produce chargedions. It is preferred that the ionization conditions are such thatprimarily a singly charged parent ion is produced. A first quadrupole,Q0, is operated in radio frequency (RF) mode only and acts as an ionguide for all charged particles. The second quadrupole, Q1, is operatedin RF+DC mode to pass only a narrow range of mass-to-charge ratios (thatincludes the mass-to-charge ratio of the reporter signals). Thisquadrupole selects the mass-to-charge ratio of interest. Quadrupole Q2,surrounded by a collision cell, is operated in RF only mode and acts asion guide. The collision cell surrounding Q2 will be filled toappropriate pressure with a gas to fracture the input ions bycollisionally induced dissociation. The collision gas preferably ischemically inert, but reactive gases can also be used. Preferredmolecular systems utilize reporter signals that contain scissile bonds,labile bonds, or combinations, such that these bonds will bepreferentially fractured in the Q2 collision cell.

A. Reporter Molecule Labeling

In one form of the disclosed method, referred to as reporter moleculelabeling (RML), reporter signals are associated with analytes to bedetected and/or quantitated. For example, a reporter signal can beassociated with a specific binding molecule that interacts with theanalyte of interest. Such a combination is referred to as a reportermolecule. The specific binding molecule in the reporter moleculeinteracts with the analyte thus associating the reporter signal with theanalyte. Alternatively, a reporter signal can be associated with ananalyte indirectly. In this mode, a “coding” molecule containing aspecific binding molecule and a coding tag is associated with theanalyte (via the specific binding molecule). Alternatively, a coding tagcan be coupled or directly associated with the analyte. Then a reportersignal associated with a decoding tag (such a combination is anotherform of reporter molecule) is associated with the coding moleculethrough an interaction between the coding tag and the decoding tag. Anexample of this interaction is hybridization where the coding anddecoding tags are complementary nucleic acid sequences. The result is anindirect association of the reporter signal with the analyte. This modehas the advantage that all of the interactions of the reporter signalswith the coding molecule can be made chemically and physically similarby using the same types of coding tags and decoding tags for all of thecoding molecules and reporter molecules in a set.

B. Reporter Signal Labeling

In another form of the disclosed method, referred to as reporter signallabeling (RSL), reporter signals are used for sensitive detection of oneor multiple analytes. In the method, analytes labeled with reportersignals are analyzed using the reporter signals to distinguish thelabeled analytes (where the analytes are labeled with the reportersignals). Detection of the reporter signals indicates the presence ofthe corresponding analyte(s). The detected analyte(s) can then beanalyzed using known techniques. The labels provide a uniqueanalyte/label composition that can specifically identify the analyte(s).This is accomplished through the use of the reporter signals as thelabels. The labeled analyte(s) can be fragmented prior to analysis. Ananalyte sample to be analyzed can also be subjected to fractionation orseparation to reduce the complexity of the samples. Fragmentation andfractionation can also be used together in the same assay. Suchfragmentation and fractionation can simplify and extend the analysis ofthe analytes.

Reporter signals can be coupled or directly associated with an analyte.For example, a reporter signal can be coupled to an analyte via reactivegroups, or a reporter molecule (composed of a specific binding moleculeand a reporter signal) can be associated with an analyte. The reportersignals can be attached to analytes in any manner. For example, reportersignals can be covalently coupled to proteins through a sulfur-sulfurbond between a cysteine on the protein and a cysteine on the reportersignal. Many other chemistries and techniques for coupling compounds toanalytes are known and can be used to couple reporter signals toanalytes. For example, coupling can be made using thiols, epoxides,nitrites for thiols, NHS esters, isothiocyanates for amines, andalcohols for carboxylic acids. Reporter signals can be attached toanalytes either directly or indirectly, for example, via a linker.

Reporter signals, or constructs containing reporters signals, also canbe attached or coupled to analytes by ligation. Methods for ligation ofnucleic acids are well known (see, for example, Sambrook et al.Molecular Cloning: A Laboratory Manual, second edition, 1989, ColdSpring Harbor Laboratory Press, New York.), and efficient proteinligation is known (see, for example, Dawson et al., “Synthesis ofproteins by native chemical ligation” Science 266, 776-9 (1994); Hackenget al., “Chemical synthesis and spontaneous folding of a multidomainprotein: anticoagulant microprotein S” Proc Natl Acad Sci USA 97:14074-8(2000); Dawson et al., “Synthesis of Native Proteins by ChemicalLigation” Ann. Rev. Biochem. 69:923-960 (2000); U.S. Pat. No. 6,184,344;PCT Publication WO 98/28434).

Alternatively, a reporter signal can be associated with an analyteindirectly. In this mode, a “coding” molecule containing a specificbinding molecule and a coding tag can be associated with the analyte(via the specific binding molecule). Alternatively, a coding tag can becoupled or directly associated with the analyte. Then a reporter signalassociated with a decoding tag (such a combination is another form ofreporter molecule) is associated with the coding molecule through aninteraction between the coding tag and the decoding tag. An example ofthis interaction is hybridization where the coding and decoding tags arecomplementary nucleic acid sequences. The result is an indirectassociation of the reporter signal with the analyte. This mode has theadvantage that all of the interactions of the reporter signals with thecoding molecule can be made chemically and physically similar by usingthe same types of coding tags and decoding tags for all of the codingmolecules and reporter molecules in a set.

Reporter signals can be fragmented, decomposed, reacted, derivatized, orotherwise modified, preferably in a characteristic way. This allows ananalyte to which the reporter signal is attached to be identified by thecorrelated detection of the labeled analyte and one or more of theproducts of the labeled analyte following fragmentation, decomposition,reaction, derivatization, or other modification of the reporter signal(the labeled analyte is the analyte/reporter signal combination). Thealteration of the reporter signal will alter the labeled analyte in acharacteristic and detectable way. Together, the detection of acharacteristic labeled analyte and a characteristic product of thelabeled analyte can uniquely identify the analyte. In this way, usingthe disclosed method and materials, one or more analytes can bedetected, either alone or together (for example, in a multiplex assay).Further, one or more analytes in one or more samples can be detected ina multiplex manner. Preferably, for mass spectrometry reporter signals,the reporter signals are fragmented to yield fragments of similar chargebut different mass.

Preferably, the reporter signals are fragmented to yield fragments ofsimilar charge but different mass. This allows each labeled analyte(and/or each reporter signal) in a set to be distinguished by thedifferent mass-to-charge ratios of the fragments of the reportersignals. This is possible since, although the unfragmented reportersignals in a set are isobaric, the fragments of the different reportersignals are not. In the disclosed method, this allows eachanalyte/reporter signal combination to be distinguished by themass-to-charge ratios of the analyte/reporter signals afterfragmentation of the reporter signal.

In preferred embodiments, reporter signals are used in sets where allthe reporter signals in the set have similar properties (such as similarmass-to-charge ratios). The similar properties allow the reportersignals to be distinguished and/or separated from other moleculeslacking one or more of the properties. Preferably, the reporter signalsin a set have the same mass-to-charge ratio (m/z). That is, the reportersignals in a set are isobaric. This allows the reporter signals (or anyanalytes to which they are attached) to be separated precisely fromother molecules based on mass-to-charge ratio. The result of thefiltering is a huge increase in the signal to noise ratio (S/N) for thesystem, allowing more sensitive and accurate detection. Alternatively,or in addition, reporter signals can be used in sets such that theresulting labeled analytes will have similar properties allowing thelabeled analytes to be distinguished and/or separated from othermolecules lacking one or more of the properties.

Analytes can be detected using the disclosed reporter signals in avariety of ways. For example, the analyte and attached reporter signalcan be detected together, one or more fragments of the analyte and theattached reporter signal(s) can be detected together, the fragments ofthe reporter signal can be detected, or a combination. Preferreddetection involves detection of the analyte/reporter signal both beforeand after fragmentation of the reporter signal.

A preferred form of the disclosed method involves correlated detectionof the reporter signals both before and after fragmentation of thereporter signal. This allows labeled analytes to be detected andidentified via the change in labeled analyte. That is, the nature of thereporter signal detected (non-fragmented versus fragmented) identifiesthe analyte as labeled. Where the analytes and reporter signals aredetected by mass-to-charge ratio, the change in mass-to-charge ratiobetween fragmented and non-fragmented samples provides the basis forcomparison. Such mass-to-charge ratio detection is preferablyaccomplished with mass spectrometry.

As an example, an analyte in a sample can be labeled with reportersignal designed as a mass spectrometry label. The labeled analyte can besubjected to mass spectrometry. A peak corresponding to theanalyte/reporter signal will be detected. Fragmentation of the reportersignal in the mass spectrometer (preferably in a collision cell) wouldresult in a shift in the peak corresponding to the loss of a portion ofthe attached reporter signal, the appearance of a peak corresponding tothe lost fragment, or a combination of both events. Significantly, theshift observed will depend on which reporter signal is on the analytesince different reporter signals will, by design, produce fragments withdifferent mass-to-charge ratios. The combination event of detection ofthe parent mass-to-charge (with no collision gas) and the mass-to-chargecorresponding to the loss of the fragment from the reporter signal (withcollision gas) indicates a labeled analyte. The identity of the analytecan be determined by standard mass spectrometry techniques, such ascompositional analysis.

A powerful form of the disclosed method is use of analytes labeled withreporter signals to assay multiple samples (for example, time seriesassays or other comparative analyses). Knowledge of the temporalresponse of a biological system following perturbation is a verypowerful process in the pursuit of understanding the system. To followthe temporal response, a sample of the system is obtained (for example,cells from a cell culture, mice initially synchronized and sacrificed)at determined times following the perturbation. Knowledge of spatialanalyte profiles (for example, relative position within a tissuesection) is a very powerful process in the pursuit of understanding thebiological system.

In the disclosed method a series of samples can each be labeled with adifferent reporter signal from a set of reporter signals. Preferredreporter signals for this purpose would be those using differentiallydistributed mass. In particular, the use of stable isotopes is preferredto ensure that members of the set of reporter signals would behavechemically identically and yet would be distinguishable.

The labeled analytes are preferably detected using mass spectrometrywhich allows sensitive distinctions between molecules based on theirmass-to-charge ratios. The disclosed reporter signals can be used asgeneral labels in myriad labeling and/or detection techniques. A set ofisobaric reporter signals can be used for multiplex labeling and/ordetection of many analytes since the reporter signal fragments can bedesigned to have a large range of masses, with each mass individuallydistinguishable upon detection. Where the same analyte or type ofanalyte is labeled with a set of isobaric reporter signals (by, forexample, labeling the same analyte in different samples), the set oflabeled analytes that results from use of an isobaric set of reportersignals will also be isobaric. Fragmentation of the reporter signalswill split the set of labeled analytes into individually detectablelabeled proteins of characteristically different mass.

A preferred form of the disclosed method involves filtering of isobaricreporter signals (and the attached analytes) from other molecules basedon mass-to-charge ratio, fragmentation of the reporter signals toproduce fragments having different masses, and detection of thedifferent fragments based on their mass-to-charge ratios. This form ofthe method is best carried out using a tandem mass spectrometer asdescribed elsewhere herein.

The disclosed method can be used in many modes. For example, thedisclosed method can be used to detect a specific analyte (in a specificsample or in multiple samples) or multiple analytes (in a single sampleor multiple samples). In each case, the analyte(s) to be detected can beseparated either from other, unlabeled analytes or from other moleculeslacking a property of the labeled analyte(s) to be detected. Forexample, analytes in a sample can be generally labeled with reportersignals and some analytes can be separated on the basis of some propertyof the analytes. For example, the separated analytes could have acertain mass-to-charge ratio (separation based on mass-to-charge ratiowill select both labeled and unlabeled analytes having the selectedmass-to-charge ratio). As another example, all of the labeled analytescan be distinguished and/or separated from unlabeled molecules based ona feature of the reporter signal such as an affinity tag. Wheredifferent affinity tags are used, some labeled analytes can bedistinguished and/or separated from others. Reporter signal labelingallows profiling of analytes and cataloging of analytes.

In one mode of the disclosed method, multiple analytes in multiplesamples are labeled where all of the analytes in a given sample arelabeled with the same reporter signal. That is, the reporter signal isused as a general label of the analytes in a sample. Each sample,however, uses a different reporter signal. This allows samples as awhole to be compared with each other. By additionally separating ordistinguishing different analytes in the samples, one can easily analyzemany analytes in many samples in a single assay. For example, proteinsin multiple samples can be labeled with reporter signals as describedabove, and the samples mixed together. If some or all of the variouslabeled proteins are separated by, for example, association of theproteins with antibodies on an array, the presence and amount of a givenprotein in each of the samples can be determined by identifying thereporter signals present at each array element. If the proteincorresponding to a given array element was present in a particularsample, then some of the protein associated with that array element willbe labeled with the reporter signal used to label that particularsample. Detection of that reporter signal will indicate this. This samerelationship holds true for all of the other samples. Further, theamount of reporter signal detected can indicate the amount of a givenprotein in a given sample, and the simultaneous quantitation of proteinin multiple samples can provide a particularly accurate comparison ofthe levels of the proteins in the various samples.

1. Reporter Signal Protein Labeling

In one form of reporter signal labeling, referred to as reporter signalprotein labeling (RSPL), reporter signals are used for sensitivedetection of one or multiple proteins. In the method, proteins labeledwith reporter signals are analyzed using the reporter signals todistinguish the labeled proteins. Detection of the reporter signalsindicates the presence of the corresponding protein(s). The detectedprotein(s) can then be analyzed using known techniques. The labelsprovide a unique protein/label composition that can specificallyidentify the protein(s). This is accomplished through the use of thespecialized reporter signals as the labels.

Although reference is made above and elsewhere herein to detection of a“protein” or “proteins,” the disclosed method and compositions encompassproteins, peptides, and fragments of proteins or peptides. Thus,reference to a protein herein is intended to refer to proteins,peptides, and fragments of proteins or peptides unless the contextclearly indicates otherwise. As used herein “labeled protein” refers toa protein, peptide, or fragment of a protein or peptide to which areporter signal is attached unless the context clearly indicatesotherwise. The labeled protein(s) can be fragmented, such as by proteasedigestion, prior to analysis. A protein sample to be analyzed can alsobe subjected to fractionation or separation to reduce the complexity ofthe samples. Fragmentation and fractionation can also be used togetherin the same assay. Such fragmentation and fractionation can simplify andextend the analysis of the proteins.

The reporter signals can be attached to proteins in any manner. Forexample, reporter signals can be covalently coupled to proteins througha sulfur-sulfur bond between a cysteine on the protein and a cysteine onthe reporter signal. Many other chemistries and techniques for couplingcompounds to proteins are known and can be used to couple reportersignals to proteins. For example, coupling can be made using thiols,epoxides, nitriles for thiols, NHS esters, isothiocyanates for amines,and alcohols for carboxylic acids. Reporter signals can be attached toproteins either directly or indirectly, for example, via a linker.Reporter signals also can be attached to proteins by ligation (forexample, protein ligation of a reporter signal peptide to a protein).

It is possible to form labeled proteins where the reporter signal isspecifically attached to phosphopeptides. Chemistry for specificderivatization of phosphoserine or phosphotyrosine residues has beendescribed (Zhou et al. A systematic approach to the problem of proteinphosphorylation., Nat. Biotech. 19:375-378 (2001), Oda et al.,Enrichment analysis of phosphorylated proteins as a tool for probing thephosphoproteome., Nat. Biotech. 19:379-382 (2001)). Tryptic peptidestreated according to either of these two protocols will display reactivesulfhydryls at sites of protein phosphorylation. These sites may bereacted with reporter signals to generate a labeled protein.Non-phosphorylated peptides will not be derivatized.

Reporter signals can be fragmented, decomposed, reacted, derivatized, orotherwise modified, preferably in a characteristic way. This allows aprotein to which the reporter signal is attached to be identified by thecorrelated detection of the labeled protein and one or more of theproducts of the labeled protein following fragmentation, decomposition,reaction, derivatization, or other modification of the reporter signal(the labeled protein is the protein/reporter signal combination). Thealteration of the reporter signal will alter the labeled protein in acharacteristic and detectable way.

Together, the detection of a characteristic labeled protein and acharacteristic product of the labeled protein can uniquely identify theprotein. In this way, using the disclosed method and materials, one ormore proteins can be detected, either alone or together (for example, ina multiplex assay). Further, one or more proteins in one or more samplescan be detected in a multiplex manner. Preferably, for mass spectrometryreporter signals, the reporter signals are fragmented to yield fragmentsof similar charge but different mass.

Preferably, the reporter signals are fragmented to yield fragments ofsimilar charge but different mass. This allows each labeled protein(and/or each reporter signal) in a set to be distinguished by thedifferent mass-to-charge ratios of the fragments of the reportersignals. This is possible since, although the unfragmented reportersignals in a set are isobaric, the fragments of the different reportersignals are not. In the disclosed method, this allows eachprotein/reporter signal combination to be distinguished by themass-to-charge ratios of the protein/reporter signals afterfragmentation of the reporter signal.

In preferred embodiments, reporter signals are used in sets where allthe reporter signals in the set have similar properties (such as similarmass-to-charge ratios). The similar properties allow the reportersignals to be distinguished and/or separated from other moleculeslacking one or more of the properties. Preferably, the reporter signalsin a set have the same mass-to-charge ratio (m/z). That is, the reportersignals in a set are isobaric. This allows the reporter signals (or anyproteins to which they are attached) to be separated precisely fromother molecules based on mass-to-charge ratio. The result of thefiltering is a huge increase in the signal to noise ratio (S/N) for thesystem, allowing more sensitive and accurate detection. Alternatively,or in addition, reporter signals can be used in sets such that theresulting labeled proteins will have similar properties allowing thelabeled proteins to be distinguished and/or separated from othermolecules lacking one or more of the properties.

Proteins can be detected using the disclosed reporter signals in avariety of ways. For example, the protein and attached reporter signalcan be detected together, one or more peptides of the protein and theattached reporter signal(s) can be detected together, the fragments ofthe reporter signal can be detected, or a combination. Preferreddetection involves detection of the protein/reporter signal orpeptide/reporter signal both before and after fragmentation of thereporter signal.

A preferred form of the disclosed method involves correlated detectionof the reporter signals both before and after fragmentation of thereporter signal. This allows labeled proteins to be detected andidentified via the change in labeled protein. That is, the nature of thereporter signal detected (non-fragmented versus fragmented) identifiesthe protein as labeled. Where the proteins and reporter signals aredetected by mass-to-charge ratio, the change in mass-to-charge ratiobetween fragmented and non-fragmented samples provides the basis forcomparison. Such mass-to-charge ratio detection is preferablyaccomplished with mass spectrometry.

As an example, a protein in a sample can be labeled with reporter signaldesigned as a mass spectrometry label. The labeled protein can besubjected to tryptic digest followed by mass spectrometry of theresulting materials. A peak corresponding to the tryptic fragmentcontaining the reporter signal will be detected. Fragmentation of thereporter signal in the mass spectrometer (preferably in a collisioncell) would result in a shift in the peak corresponding to the loss of aportion of the attached reporter signal, the appearance of a peakcorresponding to the lost fragment, or a combination of both events.Significantly, the shift observed will depend on which reporter signalis on the protein since different reporter signals will, by design,produce fragments with different mass-to-charge ratios. The combinationevent of detection of the parent mass-to-charge (with no collision gas)and the mass-to-charge corresponding to the loss of the fragment fromthe reporter signal (with collision gas) indicates a labeled protein.The combination event may be carried out in an analogous fashion to thedetection of phosphorylation sites described above. The identity of thetryptic fragment of the protein can be determined by standard massspectrometry techniques, such as compositional analysis and peptidesequencing.

Not all labeled protein fragments that can be made in the disclosedmethod from a protein sample will be unique. Because some proteins havecommon motifs that may be identical in different proteins, some proteinfragments or peptides produced from a sample will be identical althoughthey were derived from different proteins. For example, some families ofrelated proteins have such common motifs or common amino acid sequences.Thus, in some embodiments of the disclosed method, detection of acharacteristic labeled protein may be the result of detection of acommon portion of related proteins. Such a result can be an advantagewhen detection of the family of proteins is desired. Alternatively, suchcollective detection of related proteins can be avoided by focusing ondetection of unique fragments (that is, non-identical portions) of theproteins in the family. For convenience, as used herein, detection of acommon portion of multiple related proteins is intended to beencompassed by reference to detection of a unique protein, labeledprotein, or other component, unless the context clearly indicatesotherwise.

A powerful form of the disclosed method is use of proteins labeled withreporter signals to assay multiple samples (for example, time seriesassays or other comparative analyses). Knowledge of the temporalresponse of a biological system following perturbation is a verypowerful process in the pursuit of understanding the system. To followthe temporal response a sample of the system is obtained (for example,cells from a cell culture, mice initially synchronized and sacrificed)at determined times following the perturbation. Knowledge of spatialprotein profiles (for example, relative position within a tissuesection) is a very powerful process in the pursuit of understanding thebiological system.

In the disclosed method a series of samples can each be labeled with adifferent reporter signal from a set of reporter signals. Preferredreporter signals for this purpose would be those using differentiallydistributed mass. In particular, the use of stable isotopes is preferredto ensure that members of the set of reporter signals would behavechemically identically and yet would be distinguishable. An exemplaryset of labels could be as shown in Table 1, where each of five timepoints could be labeled with one of the five indicated labels and themixture of the samples could be read out simultaneously. Theunfragmented labels are SEQ ID NO:1 and the fragmented labels are aminoacids 7-12 of SEQ ID NO:1. TABLE 1 Fragment Mass Fragment mass Sequence(amu) Sequence (amu) CG*G*G*G*DPGGGGR 949 PGGGGR 499 CG*G*G*GDPGGGG*R949 PGGGG*R 500 CG*G*GGDPGGG*G*R 949 PGGG*G*R 501 CG*GGGDPGG*G*G*R 949PGG*G*G*R 502 CGGGGDPG*G*G*G*R 949 PG*G*G*G*R 503

The labeled proteins are preferably detected using mass spectrometrywhich allows sensitive distinctions between molecules based on theirmass-to-charge ratios. The disclosed reporter signals can be used asgeneral labels in myriad labeling and/or detection techniques. A set ofisobaric reporter signals can be used for multiplex labeling and/ordetection of many proteins since the reporter signal fragments can bedesigned to have a large range of masses, with each mass individuallydistinguishable upon detection. Where the same protein or type ofprotein is labeled with a set of isobaric reporter signals (by, forexample, labeling the same protein in different samples), the set oflabeled proteins that results from use of an isobaric set of reportersignals will also be isobaric. Fragmentation of the reporter signalswill split the set of labeled proteins into individually detectablelabeled proteins of characteristically different mass.

A preferred form of the disclosed method involves filtering of isobaricreporter signals (and the attached proteins) from other molecules basedon mass-to-charge ratio, fragmentation of the reporter signals toproduce fragments having different masses, and detection of thedifferent fragments based on their mass-to-charge ratios. The method isbest carried out using a tandem mass spectrometer as described elsewhereherein.

The method allows detection of proteins, peptides and protein fragmentswhere detection provides some information on the sequence or otherstructure of the protein or peptide detected. For example, the mass ormass-to-charge ratio, the amino acid composition, or amino acid sequencecan be determined. The set of proteins, peptides and/or proteinfragments detected in a sample using particular reporter signals willproduce characteristic sets of protein and peptide information. Themethod allows a complex sample of proteins to be cataloged quickly andeasily in a reproducible manner. The disclosed method also shouldproduce two “signals” for each protein, peptide, or peptide fragment inthe sample: the original labeled protein and the altered form of thelabeled protein. This can allow comparisons and validation of a set ofdetected proteins and peptides.

Reporter signal protein labeling allows profiling of proteins, de novodiscovery of proteins, and cataloging of proteins. The method hasadvantageous properties which can be used as a detection and analysissystem for protein analysis, proteome analysis, proteomic, proteinexpression profiling, de novo protein discovery, functional genomics,and protein detection.

C. Reporter Signal Calibration

In another form of the method, referred to as reporter signalcalibration (RSC), a form of reporter signals referred to as reportersignal calibrators are mixed with analytes or analyte fragments, thereporter signal calibrators and the analytes or analyte fragments arealtered, and the altered forms of the reporter signal calibrators andaltered forms of the analytes or analyte fragments are detected.Reporter signal calibrators are useful as standards for assessing theamount of analytes present. That is, one can add a known amount of areporter signal calibrator in order to assess the amount of analytepresent comparing the amount of altered analyte or analyte fragmentdetected with the amount of altered reporter signal calibrator detectedand calibrating these amounts with the known amount of reporter signalcalibrator added (and thus the predicted amount of altered reportersignal calibrator).

The disclosed reporter signal calibration method generates, with highsensitivity, unique protein signatures related to the relative abundanceof different proteins in tissue, microorganisms, or any other biologicalsample. The disclosed method allows one to define the status of a cellor tissue by identifying and measuring the relative concentrations of asmall but highly informative subset of proteins. Such as measurement isknown as a protein signature. Protein signatures are useful, forexample, in the diagnosis, grading, and staging of cancer, in drugscreening, and in toxicity testing.

In some embodiments, each analyte or analyte fragment can share one ormore common properties with at least one reporter signal calibrator suchthat the reporter signal calibrators and analytes or analyte fragmentshaving the common property can be distinguished and/or separated fromother molecules lacking the common property.

In some embodiments, reporter signal calibrators and analytes andanalyte fragments can be altered such that the altered form of ananalyte or analyte fragment can be distinguished from the altered formof the reporter signal calibrator with which the analyte or analytefragment shares a common property. In some embodiments, the alteredforms of different reporter signal calibrators can be distinguished fromeach other. In some embodiments, the altered forms of different analytesor analyte fragments can be distinguished from each other.

In some embodiments of reporter signal calibration, the analyte oranalyte fragment is not altered and so the altered reporter signalcalibrators and the analytes or analyte fragments are detected. In thiscase, the analyte or analyte fragment can be distinguished from thealtered form of the reporter signal calibrator with which the analyte oranalyte fragment shares a common property.

In some embodiments the analyte or analyte fragment may be a reportersignal or a fragment of a reporter signal. In this case, the reportersignal calibrators serve as calibrators for the amount of reportersignal detected.

Reporter signal calibration is preferably used in connection withproteins and peptides (as the analytes). This form of reporter signalcalibration is referred to as reporter signal protein calibration.Reporter signal protein calibration is useful, for example, forproducing protein signatures of protein samples. As used herein, aprotein signature is the presence, absence, amount, or presence andamount of a set of proteins or protein surrogates.

In some embodiments of reporter signal protein calibration, the presenceof labile, scissile, or cleavable bonds in the proteins to be detectedcan be exploited. Peptides, proteins, or protein fragments (collectivelyreferred to, for convenience, as protein fragments in the remainingdescription) containing such bonds can be altered by fragmentation atthe bond. In this way, reporter signal calibrators having a commonproperty (such as mass-to-charge ratio) with the protein fragments canbe used and the altered forms of the reporter signal calibrators and thealtered (that is, fragmented) forms of the protein fragments can bedetected and distinguished. In this regard, although the proteinfragments share a common property with their matching reporter signalcalibrators, the altered forms of the reporter signal calibrators andaltered forms of protein fragments can be distinguished (because, forexample, the altered forms have different properties, such as differentmass-to-charge ratios).

As an example of reporter signal protein calibration, a protein sampleof interest can be digested with a serine protease, preferably trypsin.The digest generates a complex mixture of protein fragments. Among theseprotein fragments, there will exist a subset (approximately one proteinfragment among every 400) that contains the dipeptide Asp-Pro. Thisdipeptide sequence is uniquely sensitive to fragmentation during massspectrometry, an thus produces a high yield of ions in fragmentationmode. Since the human proteome consists of at least 2,000,000 distincttryptic peptides, the number of protein fragments containing the Asp-Prosequence is of the order of 5,000. Since some of these may exist asphosphopeptides or other modified forms, the number may be even higher.This number is sufficiently high to permit the selection of a subset(perhaps 50 to 100 or so) of fragmentable protein fragments that issuitable for generating a highly informative protein signature. Peptidesthat contain the Asp-Pro dipeptide sequence, peptides that contain aminoacids that are modified by phosphorylation inside the cell, or peptidesthat contain an internal methionine are particularly preferred for usein reporter signal calibration. Alternatively, tryptic peptidesterminating in arginine may be modified by reaction with acetylacetone(pentane-2,4-dione) to increase the frequency of fragment ions (Dikleret al., J Mass Spectrom 32:1337-49 (1997)). Selection of the subsets ofprotein fragments can be performed using bioinformatics in order tomaximize the information content of the protein signatures.

For this form of reporter signal protein calibration, the protein digestcan be mixed with a specially designed set of reporter signalcalibrators, and then is analyzed using tandem mass spectrometry. In thecase of a tandem in space instrument (for example, Q-Tof™ fromMicromass), using first quadrupole settings for single-ion filtering(defined by the molecular mass of each unique fragment, which can beobtained from sequence data), followed by a collision stage for ionfragmentation, and finally TOF spectrometry of the peptide fragments(generated by cleavage at fragile bonds, such as Asp-Pro, bondsinvolving a phosphorylated amino acid, or bonds adjacent to an oxidizedamino-acid such as methionine sulfoxide, Smith et al., Free Radic Res.26:103-11 (1997)) that arise from the original single-ion. In the secondstage, signal to noise of the TOF measurement is much larger than in aconventional MS experiment. In general, one reporter signal calibratorcan be used for each protein fragment in the sample that will be used tomake up the protein signature (such protein fragments are referred to assignature protein fragments), and each is designed to fragment in aneasily detectable pattern of masses, distinct from the fragment massesof the unfragmented signature protein fragments. The quadrupolefiltering settings are then varied in sequence over a range of values(fifty, for example), corresponding to the masses of each of the proteinfragments previously chosen to comprise the protein signature (that is,the signature protein fragments). At each filtered mass setting, therewill be two types of signals detectable by TOF after fragmentation, oneset derived from the tryptic peptide (that is, the original proteinfragment), and another set corresponding to the reporter signalcalibrator. The reporter signal calibrator permits one to calculaterelative abundance for each of the signature protein fragments. Theserelative abundance ratios, determined for a given sample, constitute theprotein signature. The detection limit of the tandem mass spectrometerin MS/MS mode, is remarkably good, perhaps of the order of 500 moleculesof peptide. This level of detection is approximately 1,000 times betterthan that for MALDI-TOF mass spectrometry, and should permit thegeneration of protein signatures from single cells.

As can be seen, for this form of reporter signal calibration, theavailability of the sequence of the entire human genome, as well as thegenomes of many other organisms, can facilitate the identification ofprotein fragments that are unique in the context of all known proteins.That is, the sequence information can be used to identify peptides thatwill be generated in a protein signature and guide selection of reportersignal calibrators.

D. Reporter Signal Fusions

In another form of the disclosed method and compositions, referred to asreporter signal fusions (RSF), reporter signal peptides are joined witha protein or peptide of interest in a single amino acid segment. Suchfusions of proteins and peptides of interest with reporter signalpeptides can be expressed as a fusion protein or peptide from a nucleicacid molecule encoding the amino acid segment that constitutes thefusion. The fusion protein or peptide is referred to herein as areporter signal fusion. The reporter signal peptides, a form of reportersignal, allow for sensitive monitoring and detection of the proteins andpeptides to which they are fused. In particular, the reporter signalfusions allow sensitive and multiplex detection of expression ofparticular proteins and peptides of interest, and/or of the genes,vectors, and expression constructs encoding the proteins and peptides ofinterest. The disclosed reporter signal fusions can also be used for anypurpose including as a source of reporter signals for other forms of thedisclosed method and compositions.

The disclosed reporter signal fusions also are useful for creatingcells, cell lines, and organisms that have particular protein(s),gene(s), vector(s), and/or expression sequence(s) labeled (that is,associated with or involved in) reporter signal fusions. For example, aset of nucleic acid constructs, each encoding a reporter signal fusionwith a different reporter signal peptide, can be used to uniquely labela set of cells, cell lines, and/or organisms. Processing of a samplefrom any of the labeled sources can result in a unique altered form ofthe reporter signal peptide (or the amino acid segment or an amino acidsubsegment) for each of the possible sources that can be distinguishedfrom the other altered forms. Detection of a particular altered formidentifies the source from which it came. As a more specific example, anucleic acid construct encoding a reporter signal fusion can beintroduced into a genetically modified plant line (for example, aRoundup resistant corn line) and the plant line can then be identifiedby detecting the reporter signal fusion. Preferred reporter signalpeptides for use in reporter signal fusions used in or associated withdifferent genes, proteins, vectors, constructs, cells, cell lines, ororganisms would be those using differentially distributed mass. Inparticular, the use of alternative amino acid sequences using the sameamino acid composition is preferred.

The disclosed reporter signal fusions also can be used to “label”particular pathways, regulatory cascades, and other suites of genes,proteins, vectors, and/or expressions sequences. Such labeling can bewithin the same cell, cell line, or organism, or across a set of cells,cell lines, or organisms. For example, nucleic acid segments encodingreporter signal fusions can be associated with endogenous expressionsequences of interest, endogenous genes of interest, exogenousexpression sequences of interest, exogenous genes of interest, or acombination. The exogenous constructs then are introduced into the cellsor organisms of interest. The association with endogenous expressionsequences or genes can be accomplished, for example, by introducing anucleic acid molecule (encoding the reporter signal fusion) forinsertion at the site of the expression sequences or gene of interest,or by creating a vector or other nucleic acid construct (containing boththe endogenous expression sequences or gene and a nucleic acid segmentencoding the reporter signal fusion) in vitro and introducing theconstruct into the cells or organisms of interest. Many other uses andmodes of use are possible, a number of which are described in theillustrations below. The disclosed reporter signal fusions can be used,for example, in any context and for any purpose that green fluorescentprotein and green fluorescent protein fusions are used. However, thedisclosed reporter signal proteins have more uses and are more usefulthan green fluorescent protein at least because of the ability tomultiplex more highly the disclosed reporter signal fusions.

Nucleic acid sequences encoding reporter signal peptides can beengineered into particular exons of a gene. This would be the normalsituation when the gene encoding the protein to be fused containsintrons, although sequence encoding a reporter signal peptide can besplit between different exons to be spliced together. Placement ofnucleic acid sequences encoding reporter signal peptides into particularexons is useful for monitoring and analyzing alternative splicing ofRNA. The appearance of a reporter signal peptide in the final proteinindicates that the exon encoding the reporter signal peptide was splicedinto the mRNA.

The reporter signal peptides can be used for sensitive detection of oneor multiple proteins (that is, the proteins to which the reporter signalpeptides are fused). In the method, proteins fused with reporter signalpeptides are analyzed using the reporter signal peptides to distinguishthe reporter signal fusions. Detection of the reporter signal peptidesindicates the presence of the corresponding protein(s). The detectedprotein(s) can then be analyzed using known techniques. The reportersignal fusions provide a unique protein/label composition that canspecifically identify the protein(s). This is accomplished through theuse of the specialized reporter signal peptides as the labels.

Although reference is made above and elsewhere herein to detection of,and fusion with, a “protein” or “proteins,” the disclosed method andcompositions encompass proteins, peptides, and fragments of proteins orpeptides. Thus, reference to a protein herein is intended to refer toproteins, peptides, and fragments of proteins or peptides unless thecontext clearly indicates otherwise. As used herein “reporter signalfusion” refers to a protein, peptide, or fragment of a protein orpeptide to which a reporter signal peptide is fused (that is, joined bypeptide bond(s) in the same polypeptide chain) unless the contextclearly indicates otherwise. The reporter signal fusion(s) can befragmented, such as by protease digestion, prior to analysis. Anexpression sample to be analyzed can also be subjected to fractionationor separation to reduce the complexity of the samples. Fragmentation andfractionation can also be used together in the same assay. Suchfragmentation and fractionation can simplify and extend the analysis ofthe expression. The reporter signal peptide(s) can be fused to a proteinin any arrangement, such as at the N-terminal end of the protein, at theC-terminal end of the protein, in or at domain junctions, or at anyother appropriate location in the protein. In some forms of the method,it is desirable that the protein remain functional. In such cases,terminal fusions or inter-domain fusions are preferable. Those of skillin the art of protein fusions generally know how to design fusions wherethe protein of interest is expected to remain functional. In otherembodiments, it is not necessary that the protein remain functional inwhich case the reporter signal peptide and protein can have any desiredstructural organization.

The reporter signal fusions can be produced by expression from nucleicacid molecules encoding the fusions. Thus, the disclosed fusionsgenerally can be designed by designing nucleic acid segments that encodeamino acid segments where the amino acid segments comprise a reportersignal peptide and a protein or peptide of interest. A given nucleicacid molecule can comprise one or more nucleic acid segments. A givennucleic acid segment can encode one or more amino acid segments. A givenamino acid segment can include one or more reporter signal peptides andone or more proteins or peptides of interest. The disclosed amino acidsegments consist of a single, contiguous polypeptide chain. Thus,although multiple amino acid segments can be part of the same contiguouspolypeptide chain, all of the components (that is, the reporter signalpeptide(s) and protein(s) and peptide(s) of interest) of a given aminoacid segment are part of the same contiguous polypeptide chain.

Reporter signal peptides can be fragmented, decomposed, reacted,derivatized, or otherwise modified, preferably in a characteristic way.This allows a protein to which the reporter signal peptide is fused tobe identified by detection of one or more of the products of thereporter signal fusion following fragmentation, decomposition, reaction,derivatization, or other modification of the reporter signal peptide.The protein can also be identified by the correlated detection of thereporter signal fusion and one or more of the products of the reportersignal fusion following fragmentation, decomposition, reaction,derivatization, or other modification of the reporter signal peptide.The alteration of the reporter signal peptide will alter the reportersignal fusion in a characteristic and detectable way. Together, thedetection of a characteristic reporter signal fusion and acharacteristic product of (that is, altered form of) the reporter signalfusion can uniquely identify the protein (although the altered formalone can be detected, if desired). In this way, using the disclosedmethod and materials, expression of one or more proteins can bedetected, either alone or together (for example, in a multiplex assay).Further, expression of one or more proteins in one or more samples canbe detected in a multiplex manner. Preferably, for mass spectrometryreporter signals, the reporter signal peptides are fragmented to yieldfragments of similar charge but different mass.

Preferably, the reporter signal peptides are fragmented to yieldfragments of similar charge but different mass. This allows eachreporter signal fusion (and/or each reporter signal peptide) in a set tobe distinguished by the different mass-to-charge ratios of the fragmentsof (that is, altered forms of) the reporter signal peptides. This ispossible since the fragments of the different reporter signal peptides(or the fragments of the reporter signal fusions) can be designed tohave different mass-to-charge ratios. In the disclosed method, thisallows each reporter signal fusion to be distinguished by themass-to-charge ratios of the reporter signal fusions after fragmentationof the reporter signal peptide.

Alteration of reporter signals peptides in reporter signal fusions canproduce a variety of altered compositions. Any or all of these alteredforms can be detected. For example, the altered form of the reportersignal peptide can be detected, the altered form of the amino acidsegment (which contains the reporter signal peptide) can be detected,the altered form of a subsegment of the amino acid segment can bedetected, or a combination of these can be detected. Where the reportersignal peptide is altered by fragmentation, the result generally will bea fragment of the reporter signal peptide and an altered form of theamino acid segment containing the protein or peptide of interest and aportion of the reporter signal peptide (that is, the portion not in thereporter signal peptide fragment).

The protein or peptide of interest also can be fragmented. The resultwould be a subsegment of the amino acid segment. The amino acidsubsegment would contain the reporter signal peptide and a portion ofthe protein or peptide of interest. When the reporter signal peptide inan amino acid subsegment is altered (which can occur before, during, orafter fragmentation of the amino acid segment), the result is an alteredform of the amino acid subsegment (and an altered form of the reportersignal peptide). This altered form of amino acid subsegment can bedetected. Where the reporter signal peptide is altered by fragmentation,the result generally will be a fragment of the reporter signal peptideand an altered form of (that is, fragment of) the amino acid subsegment.In this case, the altered form of the amino acid subsegment will containa portion of the protein or peptide of interest and a portion of thereporter signal peptide (that is, the portion not in the reporter signalpeptide fragment).

As with reporter signals generally, reporter signal peptides can be usedin sets where the reporter signal peptides in a set can have one or morecommon properties that allow the reporter signal peptides to beseparated or distinguished from molecules lacking the common property.In the case of reporter signal fusions, amino acid segments and aminoacid subsegments can be used in sets where the amino acid segments andamino acid subsegments in a set can have one or more common propertiesthat allow the amino acid segments and amino acid subsegments,respectively, to be separated or distinguished from molecules lackingthe common property. In general, the component(s) of the reporter signalfusions having common properties can depend on the component(s) to bedetected and/or the mode of the method being used.

Nucleic acid molecules encoding reporter signal fusions can be used insets where the reporter signal peptides in the reporter signal fusionsencoded by a set of nucleic acid molecules can have one or more commonproperties that allow the reporter signal peptides to be separated ordistinguished from molecules lacking the common property. Similarly,nucleic acid molecules encoding amino acid segments can be used in setswhere the reporter signal peptides in the amino acid segments encoded bya set of nucleic acid molecules can have one or more common propertiesthat allow the reporter signal peptides to be separated or distinguishedfrom molecules lacking the common property. Nucleic acid moleculesencoding amino acid segments can be used in sets where the amino acidsegments encoded by a set of nucleic acid molecules can have one or morecommon properties that allow the amino acid segments to be separated ordistinguished from molecules lacking the common property.

Nucleic acid segments (which, generally, are part of nucleic acidmolecules) encoding reporter signal fusions can be used in sets wherethe reporter signal peptides in the reporter signal fusions encoded by aset of nucleic acid segments can have one or more common properties thatallow the reporter signal peptides to be separated or distinguished frommolecules lacking the common property. Similarly, nucleic acid segmentsencoding amino acid segments can be used in sets where the reportersignal peptides in the amino acid segments encoded by a set of nucleicacid molecules can have one or more common properties that allow thereporter signal peptides to be separated or distinguished from moleculeslacking the common property. Nucleic acid segments encoding amino acidsegments can be used in sets where the amino acid segments encoded by aset of nucleic acid molecules can have one or more common propertiesthat allow the amino acid segments to be separated or distinguished frommolecules lacking the common property. Other relationships betweenmembers of the sets of nucleic acid molecules, nucleic acid segments,amino acid segments, reporter signal peptides, and proteins of interestare contemplated.

Reporter signal fusions can include other components besides a proteinof interest and a reporter signal peptide. For example, reporter signalfusions can include epitope tags or flag peptides (see, for example,Brizzard et al. (1994) Immunoaffinity purification of FLAGepitope-tagged bacterial alkaline phosphatase using a novel monoclonalantibody and peptide elution. Biotechniques 16:730-735). Epitope tagsand flag peptides can serve as tags by which reporter signal fusions canbe manipulated. The use of epitope tags and flag peptides generally isknown and can be adapted for use in the disclosed reporter signalfusions.

In preferred embodiments, reporter signal peptides, reporter signalfusions (or amino acid segments), nucleic acid segments encodingreporter signal fusion, and/or nucleic acid molecules comprising nucleicacid segments encoding reporter signal fusions are used in sets wherethe reporter signal peptides, the reporter signal fusions, and/orsubsegments of the reporter signal fusions constituting or present inthe set have similar properties (such as similar mass-to-charge ratios).The similar properties allow the reporter signals, the reporter signalfusions, or subsegments of the reporter signal fusions to bedistinguished and/or separated from other molecules lacking one or moreof the properties. Preferably, the reporter signals, the reporter signalfusions, or subsegments of the reporter signal fusions constituting orpresent in a set have the same mass-to-charge ratio (m/z). That is, thereporter signals, the reporter signal fusions, or subsegments of thereporter signal fusions in a set are isobaric. This allows the reportersignals, the reporter signal fusions, or subsegments of the reportersignal fusions to be separated precisely from other molecules based onmass-to-charge ratio. The result of the filtering is a huge increase inthe signal to noise ratio (S/N) for the system, allowing more sensitiveand accurate detection.

Cells, cell lines, organisms, and expression of genes and proteins canbe detected using the disclosed reporter signal fusions in a variety ofways. For example, the protein and attached reporter signal peptide canbe detected together, one or more peptides of the protein and theattached reporter signal peptide(s) can be detected together, thefragments of the reporter signal peptide can be detected, or acombination. Preferred detection involves detection of the reportersignal fusion both before and after fragmentation of the reporter signalpeptide.

A preferred form of the disclosed method involves correlated detectionof the reporter signal peptides both before and after fragmentation ofthe reporter signal peptide. This allows genes, proteins, vectors, andexpression constructs “labeled” with a reporter signal peptide to bedetected and identified via the change in the reporter signal fusionand/or reporter signal peptide. That is, the nature of the reportersignal fusion or reporter signal peptide detected (non-fragmented versusfragmented) identifies the gene, protein, vector, or nucleic acidconstruct from which it was derived. Where the reporter signal fusionsand reporter signal peptides are detected by mass-to-charge ratio, thechange in mass-to-charge ratio between fragmented and non-fragmentedsamples provides the basis for comparison. Such mass-to-charge ratiodetection is preferably accomplished with mass spectrometry.

As an example, a fusion between a protein of interest and a reportersignal peptide designed as a mass spectrometry label can be expressed.The reporter signal fusion can be subjected to tryptic digest followedby mass spectrometry of the resulting materials. A peak corresponding tothe tryptic fragment containing the reporter signal peptide will bedetected. Fragmentation of the reporter signal peptide in the massspectrometer (preferably in a collision cell) would result in a shift inthe peak corresponding to the loss of a portion of the attached reportersignal peptide, the appearance of a peak corresponding to the lostfragment, or a combination of both events. Significantly, the shiftobserved will depend on which reporter signal peptide is fused to theprotein since different reporter signal peptides will, by design,produce fragments with different mass-to-charge ratios. The combinationevent of detection of the parent mass-to-charge (with no collision gas)and the mass-to-charge corresponding to the loss of the fragment fromthe reporter signal peptide (with collision gas) indicates a reportersignal fusion (thus indicating expression of the reporter signal fusionand of the gene, vector, or construct encoding it).

A powerful form of the disclosed method is use of reporter signalfusions to assay multiple samples (for example, time series assays orother comparative analyses). Knowledge of the temporal response of abiological system following perturbation is a very powerful process inthe pursuit of understanding the system. To follow the temporal responsea sample of the system is obtained (for example, cells from a cellculture, mice initially synchronized and sacrificed) at determined timesfollowing the perturbation. Knowledge of spatial protein profiles (forexample, relative position within a tissue section) is a very powerfulprocess in the pursuit of understanding the biological system.

The reporter signal fusions are preferably detected using massspectrometry which allows sensitive distinctions between molecules basedon their mass-to-charge ratios. A set of isobaric reporter signalpeptides or reporter signal fusions can be used for multiplex labelingand/or detection of the expression of many genes, proteins, vectors,expression constructs, cells, cell lines, and organisms since thereporter signal peptide fragments can be designed to have a large rangeof masses, with each mass individually distinguishable upon detection.Where the same gene, protein, vector, expression construct, cell, cellline, or organism (or the same type of gene, protein, vector, expressionconstruct, cell, cell line, or organism) is labeled with a set ofreporter signal fusions that are isobaric or that include isobaricreporter signal peptides (by, for example, “labeling” the same gene,protein, vector, expression construct, cell, cell line, or organism indifferent samples), the set of reporter signal fusions or reportersignal peptides that results will also be isobaric. Fragmentation of thereporter signal peptides will split the set of reporter signal peptidesinto individually detectable reporter signal fusion fragments andreporter signal peptide fragments of characteristically different mass.

A preferred form of the disclosed method involves filtering of isobaricreporter signal fusions or reporter signal peptides from other moleculesbased on mass-to-charge ratio, fragmentation of the reporter signalpeptides to produce fragments having different masses, and detection ofthe different fragments based on their mass-to-charge ratios. The methodis best carried out using a tandem mass spectrometer as describedelsewhere herein.

Nucleic acid sequences and segments encoding reporter signal fusions canbe expressed in any suitable manner. For example, the disclosed nucleicacid sequences and nucleic acid segments can be expressed in vitro, incells, and/or in cells in organism. Many techniques and systems forexpression of nucleic acid sequences and proteins are known and can beused with the disclosed reporter signal fusions. For example, manyexpression sequences, vector systems, transformation and transfectiontechniques, and transgenic organism production methods are known and canbe used with the disclosed reporter signal peptide method andcompositions.

For example, kits for the in vitro transcription/translation of DNAconstructs containing promoters and nucleic acid sequence to betranscribed and translated are known (for example, PROTEINscript-PRO™from Ambion, Inc. Austin Tex.; Wilkinson (1999) “Cell-Free And Happy: InVitro Translation And Transcription/Translation Systems”, The Scientist13[13]:15, Jun. 21, 1999). Such constructs can be used in the genomicDNA of an organism, in a plasmid or other vector that may be transfectedinto an organism, or in in vitro systems. For example, constructscontaining a promoter sequence and a nucleic acid sequence that,following transcription and translation, results in production of greenfluorescence protein or luciferase as a reporter/marker in in vivosystems are known (for example, Sawin and Nurse, “Identification offission yeast nuclear markers using random polypeptide fusions withgreen fluorescent protein.” Proc Natl Acad Sci USA 93(26): 15146-51(1996); Chatterjee et al., “In vivo analysis of nuclear protein trafficin mammalian cells.” Exp Cell Res 236(1):346-50 (1997); Patterson etal., “Quantitative imaging of TATA-binding protein in living yeastcells.” Yeast 14(9):813-25 (1998); Dhandayuthapani et al., “Greenfluorescent protein as a marker for gene expression and cell biology ofmycobacterial interactions with macrophages.” Mol Microbiol 17(5):901-12(1995); Kremer et al., “Green fluorescent protein as a new expressionmarker in mycobacteria.” Mol Microbiol 17(5):913-22 (1995); Reiländer etal., “Functional expression of the Aequorea victoria green fluorescentprotein in insect cells using the baculovirus expression system.”Biochem Biophys Res Commun 219(1): 14-20 (1996); Mankertz et al.,“Expression from the human occludin promoter is affected by tumornecrosis factor alpha and interferon gamma” J Cell Sci, 113:2085-90(2000); White et al., “Real-time analysis of the transcriptionalregulation of HIV and hCMV promoters in single mammalian cells” J CellSci, 108:441-55 (1995)). Green fluorescence protein, or variants, havebeen shown to be stably incorporated and not interfere with theorganism—generally GFP is larger relative to the disclosed reportersignal peptides (GFP from Aequorea Victoria is 238 amino acids in size;NCBI GI:606384), and thus the generally smaller reporter signal peptidesare less likely to disrupt an expression system to which they are added.

Techniques are known for modifying promoter regions such that theendogenous promoter is replaced with a promoter-reporter construct, forexample, where the reporter is green fluorescent protein (Patterson etal., “Quantitative imaging of TATA-binding protein in living yeastcells.” Yeast 14(9): 813-25 (1998)) or luciferase. Transcription factorconcentrations are followed by monitoring the GFP or luciferase. Thesetechniques can be used with the disclosed reporter signal fusions andreporter signal fusion constructs. Techniques are also known fortargeted knock-in of nucleic acid sequences into a gene of interest,typically under control of the endogenous promoter. Such techniques,which can be used with the disclosed method and compositions, have beenused to introduce reporter/markers of the transcription and translationof the gene where the nucleic acid was inserted. The same techniques canbe used to place the disclosed reporter signal fusions under control ofendogenous expression sequences. Alternately, non-targeted knock-ins(techniques for which are also known; Hobbs et al. “Development of abicistronic vector driven by the human polypeptide chain elongationfactor 1 alpha promoter for creation of stable mammalian cell lines thatexpress very high levels of recombinant proteins” Biochem Biophys ResCommun, 252:368-72 (1998); Kershnar et al., “Immunoaffinity purificationand functional characterization of human transcription factor IIH andRNA polymerase II from clonal cell lines that conditionally expressepitope-tagged subunits of the multiprotein complexes” J Biol Chem,273:34444-53 (1998); Wu and Chiang, “Establishment of stable cell linesexpressing potentially toxic proteins by tetracycline-regulated andepitope-tagging methods” Biotechniques 21:718-22, 724-5 (1996)) can beused to follow the level or activity of transcription factors—reportersignal peptide fusions associated with the inserted nucleic acid codecan directly indicate the transcription/translation activity.

The disclosed reporter signal fusions also can be used in the detectionand analysis of protein interactions with other proteins and molecules.For example, interaction traps for protein-protein interactions includethe well known yeast two-hybrid (Fields and Song, “A novel geneticsystem to detect protein-protein interactions” Nature 340:245-6 (1989);Uetz et al., “A comprehensive analysis of protein-protein interactionsin Saccharomyces cerevisiae” Nature 403:623-7 (2000)) and relatedsystems (Ausubel et al., Current Protocols in Molecular Biology, JohnWiley & Sons, Inc., 2001; Van Criekinge and Beyaert, “Yeast two-hybrid:state of the art” Biological Procedures Online, 2(1), 1999).Incorporation of nucleic acid sequence encoding a peptide reportersignal can be introduced into these systems, for example at a terminusof the ordinarily used LacZ selection region (LacZ selection isdescribed in, for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, second edition, 1989, Cold Spring Harbor LaboratoryPress, New York). A set of such incorporated sequences (for example, ina set of such plasmids, where each plasmid has a reporter signal codingsequence and the LacZ functionality), allows the unambiguous detectionof many interactions simultaneously rather (as many differentinteractions as reporter signals used).

In another mode of reporter signal fusions, a nucleic acid sequenceencoding a reporter signal could be added to sequence encoding theconstant (C) region of T cell and B cell receptors. The reporter signalwould appear in T or B cell receptors when that C region is spliced to aJ region following transcription.

In another mode of reporter signal fusions, referred to as reportersignal presentation, the presentation of specific antigenic peptides bymajor histocompatibility (MHC) and non-major histocompatibilitymolecules can be detected and analyzed. It is well known that proteinantigens are processed by antigen presenting cells and that smallpeptides, typically 8-12 amino acids are presented by Class I and ClassII MHC molecules for recognition by T cells. The study of specific Tcell/peptide-MHC complexes is technically challenging due variouslabeling requirements (either radioactive or fluorescence) and thecommon reliance on antibody reagents that recognize specific receptorsand/or peptide-MHC complexes.

There is a need to be able to further expand our knowledge of antigenprocessing and antigen presentation. Reporter signals that have beenengineered into specific protein antigens could provide novel insightinto this process and enable new experimental approaches. For instance,consider two viral or bacterial proteins, protein A and protein B, thatdiffer by only a few amino acids. It would be useful to know if they areprocessed and presented to immune cells (for example, T cells) with thesame efficiency. By engineering reporter signals into protein A andengineered protein B to antigen presenting cells, one could test for thepresence of the different reporter signals presented on and thusdetermine if the proteins are efficiently processed and presented. Thepresence of reporter signal A (present in protein A) but not reportersignal B (present in protein B), indicates that protein A is processedand that protein B is not. The lack of antigen processing of protein Bmay then be an explanation of why a virus or bacteria escapes immunesurveillance by the immune system. Antigenic peptides are characterizedby conserved anchor residues near both the amino and carboxy ends, withmore heterogeneity tolerated in the middle. This middle heterogeneity isthus a preferred site for addition of a reporter signal peptide.

E. Rearranging Reporter Signals

Another embodiment of the disclosed method and compositions, referred toas rearranging reporter signals, enables one to detect the occurrence ofspecific gene rearrangement events, their protein products, and specificcell populations bearing those receptors. Rearranging reporter signalswill also allow one to follow the progression or development of certainreceptors and cells or populations of cells by monitoring the presenceand/or absence of a reporter signal. Design considerations forrearranged reporter signals are analogous to those required for reportersignal fusions as described elsewhere herein.

Most embodiments of the disclosed method involve intact reporter signalsthat are associated with analytes in various ways. Rearranging reportersignals make use of processes, such as biological processes, to formreporter signals by specific rearrangement of the reporter signal piecesor rearrangement of nucleic acid segments encoding only portions ofreporter signals. One form of rearranging reporter signals utilizesendogenous biological systems, such as the variable-diversity-joining(V-D-J) gene rearrangement machinery present in the mammalian immunesystem. In this system, short stretches of germline DNA (the V, D & Jgene fragments) that are not contiguous, are brought together(recombined) prior to serving as a template for transcription. Generearrangement occurs in white blood cells such as T and B lymphocytesand is a key mechanism for generating diversity of T cell and B cellantigen receptors. Theoretically, billions of different receptors can begenerated. This level of complexity makes it difficult to detect thepresence of rare rearrangement events, or receptors. PCR based assaysand flow cytometry approaches are now used to study receptor diversity.However, PCR approaches are laborious and do not provide any informationon the status of expressed protein. Flow cytometry approaches havelimited multiplexing capabilities due to emission spectra overlap of thefluorescent probes used.

If one desired to test for 50-100 T cell or B cell receptors, one wouldneed to make use of a similar number of antibodies to those receptors,something that in practice is not done. Therefore, there is a real needfor methods that would allow highly sensitive and specific detection ofspecific receptors in a highly complex pool of receptors. The ability tohighly multiplex this approach would enable currently unattainableexperimental approaches. The disclosed reporter signal technology allowslarge scale multiplexing of signals for detection.

As an example of rearranging reporter signals, transgenic mice can begenerated in which nucleic acid sequences encoding reporter signals havebeen engineered into the mouse germline. Methods for doing this are wellknown in the art and include using standard molecular biology methods toengineer rearranging reporter signal into, for example, yeast orbacterial artificial chromosomes (YACs or BACs) and then using theseconstructs to generate transgenic mice.

As an example of the use of immunoglobulin rearrangement for rearrangingreporter signals, part of a reporter signal could be encoded on the Dregion and another part of the reporter signal could be encoded on the Jregion. Upon a rearrangement event that joined the D and J regionsencoding these “partial” reporter signals, a coding sequence for a“complete” reporter signal would be generated. Following transcriptionand translation, the reporter signal would be encoded within the proteinproduct. The reporter signal could then be detected as describedelsewhere herein. In the absence of a rearrangement event that joins theengineered D and J region, no reporter signal would be detected. Byincluding sequences encoding parts of a variety of reporter signals withdifferent D and J regions, a variety of different reporter signals canbe generated by rearrangement, a different, and diagnostic, reportersignal for each of the different possible rearrangements. This systemalso could be extended to include, for example, reporter signals splitamong three or more gene regions (for example, V-D-J, V-D-D-J, etc) withthe result that multiple rearrangement events would produce the reportersignal. In this mode, the combinations of rearrangements of the reportersignal parts can give rise to an large number of different reportersignals, each characterized by the specific reporter signal partsrearranged to form the reporter signal.

Transgenic mice carrying rearranging reporter signals would enable oneto address questions that would otherwise be very difficult orimpossible to address. For instance, one could dissect what specific Tand B cell receptors (out of the thousands or millions possible) respondto specific stimuli or what cell types are present at certain stages ofdevelopment.

F. Mass Spectrometers

The disclosed methods can make use of mass spectrometers for analysis ofreporter signals, altered forms of reporters signals, and variousanalytes and analyte fragments. Mass spectrometers are generallyavailable and such instruments and their operations are known to thoseof skill in the art. Fractionation systems integrated with massspectrometers are commercially available, exemplary systems includeliquid chromatography (LC) and capillary electrophoresis (CE).

The principle components of a mass spectrometer include: (a) one or moresources, (b) one or more analyzers and/or cells, and (c) one or moredetectors. Types of sources include Electrospray Ionization (ESI) andMatrix Assisted Laser Desorption Ionization (MALDI). Types of analyzersand cells include quadrupole mass filter, hexapole collision cell, ioncyclotron trap, and Time-of-Flight (TOF). Types of detectors includeMultichannel Plates (MCP) and ion multipliers. A preferred massspectrometer for use with the disclosed method is described byKrutchinsky et al., Rapid Automatic Identification of Proteins Utilizinga Novel MALDI-Ion Trap Mass Spectrometer, Abstract of the 49^(th) ASMSConference on Mass Spectrometry and Allied Topics (May 27-31, 2001), TheRockefeller University, New York, N.Y.

Mass spectrometers with more than one analyzer/cell are known as tandemmass spectrometers. There are two types of tandem mass spectrometers, aswell as hybrids and combinations of these types: “tandem in space”spectrometers and “tandem in time” spectrometers. Tandem massspectrometers where the ions traverse more than one analyzer/cell areknown as tandem in space mass spectrometers. Tandem in spacespectrometers utilize spatially ordered elements and act upon the ionsin turn as the ions pass through each element. Tandem mass spectrometerswhere the ions remain primarily in one analyzer/cell are known as tandemin time mass spectrometers. Tandem in time spectrometers utilizetemporally ordered manipulations on the ions as the ions are containedin a space. Hybrid systems and combinations of these types are known.The ability to select a particular mass-to-charge ratio of interest in amass analyzer is typically characterized by the resolution (reported asthe centroid mass-to-charge divided by the full width at half maximum ofthe selected ions of interest). Thus resolution is an indicator of thenarrowness of the ion mass-to-charge distribution passed through theanalyzer to the detector. Reference to such resolution is generallynoted herein by referring to the ability of a mass spectrometer to passonly a narrow range of mass-to-charge ratios.

A preferred form of mass spectrometer for use in the disclosed methodsis a tandem mass spectrometer, such as a tandem in space tandem massspectrometer. As an example of the use of a tandem in space class ofinstrument, the isobaric reporter signals can be first passed through afiltering quadrupole, the reporter signals are fragmented (preferably ina collision cell), and the fragments are distinguished and detected in atime-of-flight (TOF) stage. In such an instrument the sample is ionizedin the source (for example, in a MALDI ion source) to produce chargedions. It is preferred that the ionization conditions are such thatprimarily a singly charged parent ion is produced. A first quadrupole,Q0, is operated in radio frequency (RF) mode only and acts as an ionguide for all charged particles. The second quadrupole, Q1, is operatedin RF+DC mode to pass only a narrow range of mass-to-charge ratios (thatincludes the mass-to-charge ratio of the reporter signals). Thisquadrupole selects the mass-to-charge ratio of interest. Quadrupole Q2,surrounded by a collision cell, is operated in RF only mode and acts asion guide. The collision cell surrounding Q2 can be filled toappropriate pressure with a gas to fracture the input ions bycollisionally induced dissociation when fragmentation of the reportersignals is desired. The collision gas preferably is chemically inert,but reactive gases can also be used. Preferred molecular systems utilizereporter signals that contain scissile bonds, labile bonds, orcombinations, such that these bonds will be preferentially fractured inthe Q2 collision cell.

Tandem instruments capable of MS^(N) can be used with the disclosedmethod. As an example consider; a method where one selects a set ofmolecules using a first stage filter (MS), photocleaves these moleculesto yield a set of reporter signals, selects these reporter signals usinga second stage (MS/MS), alters these reporter signals by collisionalfragmentation, detects by time of flight (MS3). Many other combinationsare possible and the disclosed method can be adapted for use with suchsystems. For example, extension to more stages, or analysis of reportersignal fragments is within the skill of those in the art.

Materials

A. Reporter Signals

Reporter signals are molecules that can be preferentially fragmented,decomposed, reacted, derivatized, or otherwise modified or altered fordetection. Detection of the modified reporter signals is preferablyaccomplished with mass spectrometry. The disclosed reporter signals arepreferably used in sets where members of a set have the samemass-to-charge ratio (m/z). This facilitates sensitive filtering orseparation of reporter signals from other molecules based onmass-to-charge ratio. Reporter signals can have any structure thatallows modification of the reporter signal and identification of thedifferent modified reporter signals. Reporter signals preferably arecomposed such that at least one preferential bond rupture can be inducedin the molecule. A set of reporter signals having nominally the samemolecular mass and arbitrarily chosen internal fragmentation points maybe constructed such that upon fragmentation each member of the set willyield unique correlated daughter fragments. For convenience, reportersignals that are fragmented, decomposed, reacted, derivatized, orotherwise modified for detection are referred to as fragmented reportersignals.

Preferred reporter signals are made up of chains of subunits such aspeptides, oligonucleotides, peptide nucleic acids, oligomers,carbohydrates, polymers, and other natural and synthetic polymers andany combination of these. Most preferred chains are peptides, and arereferred to herein as reporter signal peptides. Chains of subunits andsubunits have a relationship similar to that of a polymers and mers. Themers are connected together to form a polymer. Likewise, subunits areconnected together to form chains of subunits. Preferred reportersignals are made up of chains of similar or related subunits. These aretermed homochains or homopolymers. For example, nucleic acids are madeup of phosphonucleosides and peptides are made up of amino acids.

Reporter signals can also be made up of heterochains or heteropolymers.A heterochain is a chain or a polymer where the subunits making up thechain are different types or the mers making up the polymer aredifferent types. For example, a heterochain could be guanosine-alanine,which is made up of one nucleoside subunit and one amino acid subunit.It is understood that any combination of types of subunits can be usedwithin the disclosed compositions, sets, and methods. Any moleculehaving the required properties can be used as a reporter signal.Preferred reporter signals can be fragmented in tandem massspectrometry.

Reporter signals preferably are used in sets where all the reportersignals in the set have similar physical properties. The similar (orcommon) properties allow the reporter signals to be distinguished and/orseparated from other molecules lacking one or more of the properties.Preferably, the reporter signals in a set have the same mass-to-chargeratio (m/z). That is, the reporter signals in a set are isobaric. Thisallows the reporter signals (and/or the proteins to which they areattached) to be separated precisely from other molecules based onmass-to-charge ratio. The result of the filtering is a huge increase inthe signal to noise ratio (S/N) for the system, allowing more sensitiveand accurate detection. Sets of reporter signals can have any number ofreporter signals. For example, sets of reporter signals can have one,two or more, three or more, four or more, five or more, six or more,seven or more, eight or more, nine or more, ten or more, twenty or more,thirty or more, forty or more, fifty or more, sixty or more, seventy ormore, eighty or more, ninety or more, one hundred or more, two hundredor more, three hundred or more, four hundred or more, or five hundred ormore different reporter signals. Although specific numbers of reportersignals and specific endpoints for ranges of the number of reportersignals are recited, each and every specific number of reporter signalsand each and every specific endpoint of ranges of numbers of reportersignals are specifically contemplated, although not explicitly listed,and each and every specific number of reporter signals and each andevery specific endpoint of ranges of numbers of reporter signals arehereby specifically described.

The sets of reporter signals can be made up of reporter signals that aremade up of chains or polymers. The set of reporter signals can behomosets which means that the set is made up of one type of reportersignal or that the reporter signal is made up of homochains orhomopolymers. The set of reporter signals can also be a heteroset whichmeans that the set is made up of different reporter signals or ofreporter signals that are made up of different types of chains orpolymers. A special type of heteroset is one in which the set is made upof different homochains or homopolymers, for example one peptide chainand one nucleic acid chain. Another special type of heteroset is onewhere the chains themselves are heterochains or heteropolymers. Stillanother type of heteroset is one which is made up of bothheterochains/heteropolymers and homochains/homopolymers.

A variety of different properties can be used as the common physicalproperty used to separate reporter signals from other molecules lackingthe common property. For example, other physical properties useful ascommon properties include mass, charge, isoelectric point,hydrophobicity, chromatography characteristics, and density. It ispreferred that the physical property shared by reporter signals in a set(and used to distinguish or separate the reporter signals from othermolecules) is an overall property of the reporter signal (for example,overall mass, overall charge, isoelectric point, overall hydrophobicity,etc.) rather than the mere presence of a feature or moiety (for example,an affinity tag, such as biotin). Such properties are referred to hereinas “overall” properties (and thus, reporter signals in a set would bereferred to as sharing a “common overall property”). It should beunderstood that reporter signals can have features and moieties, such asaffinity tags, and that such features and moieties can contribute to thecommon overall property (by contributing mass, for example). However,such limited and isolated features and moieties would not serve as thesole basis of the common overall property.

A preferred common overall property is the property of subunit isomers.This property occurs when a set of at least two reporter signals (whichtypically are made up of subunit chains which are in turn made up ofsubunits, for example, like the relationship between a polymer and theunits that make up a polymer) is made up of subunit isomers, and the setcould then be called subunit isomeric or isomeric for subunits. Subunitsare discussed elsewhere herein, but reporter signals can be made up ofany type of chain, such as peptides or nucleic acids or polymer(general) which are in turn made up of subunits for example amino acidsand phosphonucleosides, and mers (general) respectively. Within eachtype of subunit there are typically multiple members that are all thesame type of subunit, but differ. For example, within the subunit type“amino acids,” there are many members, for example, ala, tyr, and ser,or any other combination of amino acids.

When a set of reporter signals is subunit isomeric or is made up ofsubunit isomers this means that each individual of the set is a subunitisomer of every other individual subunit in the set. Isomer or isomericmeans that the makeup of the subunits forming the subunit chain (i.e.,distribution or array) is the same but the overall connectivity of thesubunits, forming the chain, is different. Thus, for example, a firstreporter signal could be the chain, ala-ser-lys-gln, a second reportersignal could be the chain ala-lys-ser-gln, and a third reporter signalcould be the chain ala-ser-lys-pro. If a set of reporter signals wasmade that contained the first reporter signal and the second reportersignal, the set would be subunit isomeric because the first reportersignal and the second reporter signal have the same makeup, i.e. eachhas one ala, one ser, one lys, and one gln, but each chain has adifferent connectivity. If, however, the set of reporter signals weremade which contained the first, second, and third reporter signals theset would not be isomeric because the make up of each chain would not bethe same because the first and second chains do not have a pro and thethird chain does not have a gln.

Another illustration is the following: a first reporter signal could bethe chain, ala-guanosine-lys-adenosine, a second reporter signal couldbe the chain ala-adenosine-lys-guanosine, and a third reporter signalcould be the chain ala-ser-lys-pro. If a set of reporter signals wasmade that contained the first reporter signal and the second reportersignal, the set would be subunit isomeric because the first reportersignal and the second reporter signal have the same makeup, i.e. eachhas one ala, one guanosine, one lys, and one adenosine, but each chainhas a different connectivity. If, however, the set of reporter signalswere made which contained the first, second, and third reporter signalsthe set would not be isomeric because the makeup of each chain would notbe the same because the first and second chains do not have a pro or aser and the third chain does not have a guanosine or adenosine. Thisillustration shows that the sets can be made up of, or include,heterochains and still be considered subunit isomers.

It is preferred that the common property of reporter signals is not anaffinity tag. Nevertheless, even in such a case, reporter signals thatotherwise have a common property may also include an affinity tag—and infact may all share the same affinity tag—so long as another commonproperty is present that can be (and, in some embodiments of thedisclosed method, is) used to separate reporter signals sharing thecommon property from other molecules lacking the common property. Withthis in mind, it is preferred that, if chromatography or otherseparation techniques are used to separate reporter signals based on thecommon property, the affinity be based on an overall physical propertyof the reporter signals and not on the presence of, for example, afeature or moiety such as an affinity tag. As used herein, a commonproperty is a property shared by a set of components (such as reportersignals). That is, the components have the property “in common.” Itshould be understood that reporter signals in a set may have numerousproperties in common. However, as used herein, the common properties ofreporter signals referred to are only those used in the disclosed methodto distinguish and/or separate the reporter signals sharing the commonproperty from molecules that lack the common property.

Reporter signals in a set can be fragmented, decomposed, reacted,derivatized, or otherwise modified or altered to distinguish thedifferent reporter signals in the set. Preferably, the reporter signalsare fragmented to yield fragments of similar charge but different mass.The reporter signals can also be fragmented to yield fragments ofdifferent charge and mass. Such changes allow each reporter signal in aset to be distinguished by the different mass-to-charge ratios of thefragments of the reporter signals. This is possible since, although theunfragmented reporter signals in a set are isobaric, the fragments ofthe different reporter signals are not. Thus, a key feature of thedisclosed reporter signals is that the reporter signals have asimilarity of properties while the modified reporter signals aredistinguishable.

Differential distribution of mass in the fragments of the reportersignals can be accomplished in a number of ways. For example, reportersignals of the same nominal structure (for example, peptides having thesame amino acid sequence), can be made with different distributions ofheavy isotopes, such as deuterium (²H), tritium (³H) ¹⁷O, ¹⁸O, ¹³C, or¹⁴C; stable isotopes are preferred. All reporter signals in the setwould have the same number of a given heavy isotope, but thedistribution of these would differ for different reporter signals. Anexample of such a set of reporter signals is A*G*SLDPAGSLR,A*GSLDPAG*SLR, and AGSLDPA*G*SLR (SEQ ID NO:2), where the asteriskindicates at least one heavy isotope substituted amino acid. For asingly charged parent ion and, following fragmentation at the scissileDP bond, one predominantly charged daughter, there are threedistinguishable primary daughter ions, PAGSLR⁺, PAG*SLR⁺, PA*G*SLR⁺(amino acids 6-11 of SEQ ID NO:2).

Similarly, reporter signals of the same general structure (for example,peptides having the same amino acid sequence), can be made withdifferent distributions of modifications or substituent groups, such asmethylation, phosphorylation, sulphation, and use of seleno-methioninefor methionine. All reporter signals in the set would have the samenumber of a given modification, but the distribution of these woulddiffer for different reporter signals. An example of such a set ofreporter signals is AGS*M*LDPAGSMLR, AGS*MLDPAGSM*LR, andAGS*MLDPAGS*M*LR (SEQ ID NO:3), where S* indicates phosphoserine ratherthan serine, and, M* indicates seleno-methionine rather than methionine.For a singly charged parent ion and, following fragmentation at thescissile DP bond, one predominantly charged daughter, there are threedistinguishable primary daughter ions, PAGSMLR⁺, PAGSM*LR⁺, PAGS*M*LR⁺(amino acids 7-13 of SEQ ID NO:3).

Reporter signals of the same nominal composition (for example, made upof the same amino acids), can be made with different ordering of thesubunits or components of the reporter signal. All reporter signals inthe set would have the same number of subunits or components, but thedistribution of these would be different for different reporter signals.An example of such a set of reporter signals is AGSLADPGSLR (SEQ IDNO:4), ALSLADPGSGR (SEQ ID NO:5), ALSLGDPASGR (SEQ ID NO:6). For asingly charged parent ion and, following fragmentation at the scissileDP bond, one predominantly charged daughter, there are threedistinguishable primary daughter ions, PGSLR⁺ (amino acids 7-11 of SEQID NO:4), PGSGR⁺ (amino acids 7-11 of SEQ ID NO:5), PASGR⁺ (amino acids7-11 of SEQ ID NO:6).

Reporter signals having the same nominal composition (for example, madeup of the same amino acids), can be made with a labile or scissile bondat a different location in the reporter signal. All reporter signals inthe set would have the same number and order of subunits or components.Where the labile or scissile bond is present between particular subunitsor components, the order of subunits or components in the reportersignal can be the same except for the subunits or components creatingthe labile or scissile bond. Reporter signal peptides used in reportersignal fusions preferably use this form of differential massdistribution. An example of such a set of reporter signals isAGSLADPGSLR (SEQ ID NO:4), AGSDPLAGSLR (SEQ ID NO:7), ADPGSLAGSLR (SEQID NO:8). For a singly charged parent ion and, following fragmentationat the scissile DP bond, one predominantly charged daughter, there arethree distinguishable primary daughter ions, PGSLR⁺ (amino acids 7-11 ofSEQ ID NO:4), PLAGSLR⁺ (amino acids 5-11 of SEQ ID NO:7), PGSLAGSLR⁺(amino acids 3-11 of SEQ ID NO:8).

Each of these modes can be combined with one or more of the other modesto produce differential distribution of mass in the fragments of thereporter signals. For example, different distributions of heavy isotopescan be used in reporter signals where a labile or scissile bond isplaced in different locations. Different mass distribution can beaccomplished in other ways. For example, reporter signals can have avariety of modifications introduced at different positions. Someexamples of useful modifications include acetylation, methylation,phosphorylation, seleno-methionine rather than methionine, sulphation.Similar principles can be used to distribute charge differentially inreporter signals. Differential distribution of mass and charge can beused together in sets of reporter signals.

Reporter signals can also contain combinations of scissile bonds andlabile bonds. This allows more combinations of distinguishable signalsor to facilitate detection. For example, labile bonds may be used torelease the isobaric fragments, and the scissile bonds used to decodethe proteins.

Selenium substitution can be used to alter the mass of reporter signals.Selenium can substitute for sulfur in methionine, resulting in themodified amino acid selenomethionine. Selenium is approximately fortyseven mass units larger than sulfur. Mass spectrometry may be used toidentify peptides or proteins incorporating selenomethionine andmethionine at a particular ratio. Small proteins and peptides with knownselenium/sulfur ratio are preferably produced by chemical synthesisincorporating selenomethionine and methionine at the desired ratio.Larger proteins or peptides may be by produced from an E. coliexpression system, or any other expression system that insertsselenomethionine and methionine at the desired ratio (Hendrickson etal., Selenomethionyl proteins produced for analysis by multiwavelengthanomalous diffraction (MAD): a vehicle for direct determination ofthree-dimensional structure. Embo J, 9(5):1665-72 (1990), Cowie andCohen, Biosynthesis by Escherichia coli of active altered proteinscontaining selenium instead of sulfur. Biochimica et Biophysica Acta,26:252-261 (1957), and Oikawa et al., Metalloselenonein, the seleniumanalogue of metallothionein: synthesis and characterization of itscomplex with copper ions. Proc Natl Acad Sci USA, 88(8):3057-9 (1991).

Some forms of reporter signals can include one or more affinity tags.Such affinity tags can allow the detection, separation, sorting, orother manipulation of the labeled proteins, reporter signals, orreporter signal fragments based on the affinity tag. Such affinity tagsare separate from and in addition to (not the basis of) the commonproperties of a set of reporter signals that allows separation ofreporter signals from other molecules. Rather, such affinity tags servethe different purpose of allowing manipulation of a sample prior to oras a part of the disclosed method, not the means to separate reportersignals based on the common property. Reporter signals can have none,one, or more than one affinity tag. Where a reporter signal has multipleaffinity tags, the tags on a given reporter signal can all be the sameor can be a combination of different affinity tags. Affinity tags alsocan be used to distribute mass and/or charge differentially on reportertags following the principles described above and elsewhere herein.Affinity tags can be used with reporter signals in a manner similar tothe use of affinity labels as described in PCT Application WO 00/11208.

Peptide-DNA conjugates (Olejnik et al., Nucleic Acids Res.,27(23):4626-31 (1999)), synthesis of PNA-DNA constructs, and specialnucleotides such as the photocleavable universal nucleotides of WO00/04036 can be used as reporter signals in the disclosed method. Usefulphotocleavable linkages are also described by Marriott and Ottl,Synthesis and applications of heterobifunctional photocleavablecross-linking reagents, Methods Enzymol. 291:155-75 (1998).

Photocleavable bonds and linkages are useful in (and for use with)reporter signals because it allows precise and controlled fragmentationof the reporter signals (for subsequent detection) and precise andcontrolled release of reporter signals from analytes (or otherintermediary molecules) to which they are attached. A variety ofphotocleavable bonds and linkages are known and can be adapted for usein and with reporter signals. Recently, photocleavable amino acids havebecome commercially available. For example, an Fmoc protectedphotocleavable slightly modified phenylalanine (Fmoc-D,L-β Phe(2-NO₂))is available (Catalog Number 0011-F; Innovachem, Tucson, Ariz.). Theintroduction of the nitro group into the phenylalanine ring causes theamino acid to fragment under exposure to UV light (at a wavelength ofapproximately 350 nm). The nitrogen laser emits light at approximately337 nm and can be used for fragmentation. The wavelength used will notcause significant damage to the rest of the peptide.

Fmoc synthesis is a common technique for peptide synthesis andFmoc-derivative photocleavable amino acids can be incorporated intopeptides using this technique. Although photocleavable amino acids areusable in and with any reporter signal, they are particularly useful inpeptide reporter signals.

Use of photocleavable bonds and linkages in and with reporter signalscan be illustrated with the following examples. Materials on a blankplastic substrate (for example, a Compact Disk (CD)) may be directlymeasured from that surface using a MALDI source ion trap. For example, athin section of tissue sample, flash frozen, could be applied to the CDsurface. A reporter molecule (for example, an antibody with a reportersignal attached via a photocleavable linkage) can be applied to thetissue surface. Recognition of specific components within the tissueallows for some of the antibody/reporter signal conjugates to associate(excess conjugate is removed during subsequent wash steps). The reportersignal then can be released from the antibody by applying a UV light anddetected directly using the MALDI ion trap instrument. For example, apeptide of sequence CF*XXXXXDPXXXXXR (SEQ ID NO:24)(which contains areporter signal) can be attached to an antibody using a disulfide bondlinkage method. Exposure to the UV source of a MALDI laser will cleavethe peptide at the modified phenylalanine, F*, releasing theXXXXXDPXXXXXR reporter signal (amino acids 3-15 of SEQ ID NO:24). Thereporter signal subsequently can be fragmented at the DP bond and thecharged fragment detected as described elsewhere herein.

Another example of the use of photocleavable linkages with reportersignals involves DNA-peptide chimeras used as reporter molecules. Suchreporter molecules are useful as probes to detect particular nucleicacid sequences. In a DNA-peptide chimera (or PNA-peptide chimera), thepeptide portion can be or include a reporter signal. Placement of aphotocleavable phenylalanine, for example, near the DNA peptide junctionof the reporter molecule allows for the release of the reporter signalfrom the reporter molecule by UV light. The released reporter signal canbe detected directly or fragmented and detected as described elsewhereherein. Similarly to the case of the antibody-peptide reporter moleculedescribed above, the DNA-peptide chimera can be associated with anucleic acid molecule present on the surface of a substrate such as a CDand the reporter signal released using the UV source of a MALDI laser.

A photocleavable linkage also can be incorporated into a reporter signaland used for fragmentation of the reporter signal in the disclosedmethods. For example, a photocleavable amino acid (such as thephotocleavable phenylalanine) can be incorporated at any desiredposition in a peptide reporter signal. A reporter signal such asXXXXXXF*XXXXXR containing photocleavable phenylalanine (F*) that isphotocleavable. The reporter signal can then be fragmented using theappropriate wavelength of light and the charged fragment detected. Whenionizing the reporter signal (from a surface, for example) fordetection, a MALDI laser that does not cause significant photocleavage(for example, Er:YAG at 2.94 μm) can be used for ionization and a secondlaser (for example, Nitrogen at 337 nm) can be used to fragment thereporter signal. In this case XXXXXXFXXXXXR⁺ would be photocleaved toyield XXXXXR⁺. The second laser may intersect the reporter signal ionpacket at any location. Modification to the vacuum system of a massspectrometer for this purpose is straightforward.

The use of photocleavable linkages in reporter signals is particularlyuseful when the analyte (or other component) to which the reportersignal is attached could fragment at a scissile bond in a collisioncell. For example, in reporter signal fusions, a proteinfragment/reporter signal polypeptide could be generated that contained ascissile bond in both the protein fragment portion and the reportersignal portion. An example would be XXXXXXXXXDPXXX(XXXXXXXPXXXXXXXR)XXXX(SEQ ID NO:25), where the sequence in parenthesis indicate the reportersignal portion and the DP dipeptides contain scissile bonds. Fragmentingthis polypeptide in a collision cell could result in fragmentation ateither or both of the DP bonds, thus complicating the fragment spectrum.Use of a photocleavable linkage (such as a photocleavable amino acid) inthe reporter signal portion would allow specific photocleavage of thereporter signal during analysis. For example, an analogous polypeptideXXXXXXXXXDPXXX(XXXXXXXF*XXXXXXXR)XXXX (SEQ ID NO:26) would allowspecific photocleavage a the F* position of the reporter signal.

Multiple photocleavable bonds and/or linkages can be used in or with thesame reporter signals or reporter signal conjugates (such as reportermolecules or reporter signal fusions) to achieve a variety of effects.For example, different photocleavable linkages that are cleaved bydifferent wavelengths of light can be used in different parts ofreporter signals or reporter signal conjugates to be cleaved atdifferent stages of the method. Different fragmentation wavelengthsallow sequential processing which enables, for example, the combinationsof the release and fragmentation methods.

As an example, a peptide containing two photocleavable amino acids, Z(cleavage wavelength in the infrared) and F* (photocleavablephenylalanine, cleavage wavelength in UV) can be constructed of the formXZXXXXXXF*XXXXXXR where the amino terminus can be attached to an analyteor other molecule utilizing known chemistry. The result is a reportersignal/analyte conjugate (or, alternatively, a reporter molecule). Thereporter signal can be released from the conjugate by exposing theconjugate to an appropriate wavelength of light (infrared in thisexample), thus cleaving the bond at Z. Once the parent ion is selectedand stored in the ion trap, the reporter signal can be fragmented byexposing it to an appropriate wavelength of light (UV in this example)to produce the daughter ion (XXXXXXR⁺) which can be detected andquantitated.

Reporter signal calibrators are a special form of reporter signalcharacterized by their use in reporter signal calibration. Reportersignal calibrators can be any form of reporter signal, as describedabove and elsewhere herein, but are used as separate molecules that arenot physically associated with analytes being assessed. Thus, reportersignal calibrators need not (and preferably do not) have reactive groupsfor coupling to analytes and need not be (and preferably are not)associated with specific binding molecules or other molecules orcomponents described herein as being associated with reporter signals.

Reporter signal calibrators preferably share one or more commonproperties with one or more analytes. Reporter signal calibrators andanalytes that share one or more common properties are referred to as areporter signal calibrator/analyte set. When only one analyte and onereporter signal calibrator share the common property they also can bereferred to as a reporter signal calibrator/analyte pair. Reportersignal calibrators and analytes in a reporter signal calibrator/analyteset are said to be matching. The common property allows a reportersignal calibrator and its matching analyte to be distinguished and/orseparated from other molecules lacking one or more of the properties.Preferably, the reporter signal calibrators and analytes in a set havethe same mass-to-charge ratio (m/z). That is, the matching reportersignal calibrators and analytes in a set are isobaric. This allows thereporter signal calibrators and analytes to be separated precisely fromother molecules based on mass-to-charge ratio. Reporter signalcalibrators can be fragmented, decomposed, reacted, derivatized, orotherwise modified or altered to distinguish the altered reporter signalcalibrators from their matching analytes. The analytes can also befragmented. Preferably, the reporter signal calibrators are fragmentedto yield fragments of similar charge but different mass. The reportersignal calibrators can also be fragmented to yield fragments ofdifferent charge and mass. Such changes allow the reporter signalcalibrator to be distinguished from its matching analyte (and otheranalytes and/or reporter signal calibrators that are members of the sameset, if any) by the different mass-to-charge ratio of the fragment ofthe reporter signal calibrator. This is possible since, although theunfragmented reporter signal calibrator(s) and analyte(s) in a set areisobaric, the fragments of the reporter signal calibrator(s) are not.Thus, a key feature of the disclosed reporter signal calibrators is thatthe reporter signal calibrators have a similarity of properties withtheir matching analytes while the modified reporter signal calibratorsare distinguishable from their matching analytes.

Preferred analytes for use with reporter signal calibrators areproteins, peptides, and/or protein fragments (collectively referred tofor convenience as proteins). Reporter signal calibrators and proteinsthat share one or more common properties are referred to as a reportersignal calibrator/protein set. When only one protein and one reportersignal calibrator share the common property they also can be referred toas a reporter signal calibrator/protein pair. Reporter signalcalibrators and proteins in a reporter signal calibrator/analyte set aresaid to be matching.

As described elsewhere herein, reporter signal calibrators can be usedas standards for assessing the presence and amount of analytes insamples. For this purpose, a reporter signal calibrator designed foreach analyte to be assessed can be mixed with the sample to be analyzed.Analytes and their matching reporter signal calibrators are thenprocessed together to result in detection of both analytes and reportersignal calibrators (preferably in their altered forms). The amount ofreporter signal calibrator or altered reporter signal calibratordetected provides a standard (since the amount of reporter signalcalibrator added can be known) against which the amount of analyte oraltered analyte detected can be compared. This allows the amount ofanalyte present in the sample to be accurately gauged.

B. Analytes

The disclosed methods make use of analytes generally as objects ofdetection, measurement and/or analysis. Analytes can be any molecule orportion of a molecule that is to be detected, measured, or otherwiseanalyzed. An analyte need not be a physically separate molecule, but maybe a part of a larger molecule. Analytes include biological molecules,organic molecules, chemicals, compositions, and any other molecule orstructure to which the disclosed method can be adapted. It should beunderstood that different forms of the disclosed method are moresuitable for some types of analytes than other forms of the method.Analytes are also referred to as target molecules.

Preferred analytes are biological molecules. Biological moleculesinclude but are not limited to proteins, peptides, enzymes, amino acidmodifications, protein domains, protein motifs, nucleic acid molecules,nucleic acid sequences, DNA, RNA, mRNA, cDNA, metabolites,carbohydrates, and nucleic acid motifs. As used herein, “biologicalmolecule” and “biomolecule” refer to any molecule or portion of amolecule or multi-molecular assembly or composition, that has abiological origin, is related to a molecule or portion of a molecule ormulti-molecular assembly or composition that has a biological origin.Biomolecules can be completely artificial molecules that are related tomolecules of biological origin.

Although reference is made above and elsewhere herein to detection of a“protein” or “proteins,” the disclosed method and compositions encompassproteins, peptides, and fragments of proteins or peptides. Thus,reference to a protein herein is intended to refer to proteins,peptides, and fragments of proteins or peptides unless the contextclearly indicates otherwise.

C. Analyte Samples

Any sample from any source can be used with the disclosed method. Ingeneral, analyte samples should be samples that contain, or may contain,analytes. Examples of suitable analyte samples include cell samples,tissue samples, cell extracts, components or fractions purified fromanother sample, environmental samples, culture samples, tissue samples,bodily fluids, and biopsy samples. Numerous other sources of samples areknown or can be developed and any can be used with the disclosed method.Preferred analyte samples for use with the disclosed method are samplesof cells and tissues. Analyte samples can be complex, simple, oranywhere in between. For example, an analyte sample may include acomplex mixture of biological molecules (a tissue sample, for example),an analyte sample may be a highly purified protein preparation, or asingle type of molecule.

D. Protein Samples

Any sample from any source can be used with the disclosed method. Ingeneral, protein samples should be samples that contain, or may contain,protein molecules. Examples of suitable protein samples include cellsamples, tissue samples, cell extracts, components or fractions purifiedfrom another sample, environmental samples, biofilm samples, culturesamples, tissue samples, bodily fluids, and biopsy samples. Numerousother sources of samples are known or can be developed and any can beused with the disclosed method. Preferred protein samples for use withthe disclosed method are samples of cells and tissues. Protein samplescan be complex, simple, or anywhere in between. For example, a proteinsample may include a complex mixture of proteins (a tissue sample, forexample), a protein sample may be a highly purified protein preparation,or a single type of protein.

E. Reporter Molecules

Reporter molecules are molecules that combine a reporter signal with aspecific binding molecule or decoding tag. Preferably, the reportersignal and specific binding molecule or decoding tag are covalentlycoupled or tethered to each other. As used herein, molecules are coupledwhen they are covalent joined, directly or indirectly. One form ofindirect coupling is via a linker molecule. The reporter signal can becoupled to the specific binding molecule or decoding tag by any ofseveral established coupling reactions. For example, Hendrickson et al.,Nucleic Acids Res., 23(3):522-529 (1995) describes a suitable method forcoupling oligonucleotides to antibodies.

One form of reporter molecule has a peptide nucleic acid as the decodingtag and a reporter signal peptide as the reporter signal. The peptidenucleic acid can associate with, for example, an oligonucleotide codingtag, thus associating the reporter signal peptide with the coding tag.As described elsewhere herein, coding tags can be used to labeledanalytes and other molecules.

As used herein, a molecule is said to be tethered to another moleculewhen a loop of (or from) one of the molecules passes through a loop of(or from) the other molecule. The two molecules are not covalentlycoupled when they are tethered. Tethering can be visualized by theanalogy of a closed loop of string passing through the hole in thehandle of a mug. In general, tethering is designed to allow one or bothof the molecules to rotate freely around the loop.

F. Specific Binding Molecules

A specific binding molecule is a molecule that interacts specificallywith a particular molecule or moiety. The molecule or moiety thatinteracts specifically with a specific binding molecule is referred toherein as an analyte, such as an analyte. Preferred analytes areanalytes. It is to be understood that the term analyte refers to, bothseparate molecules and to portions of such molecules, such as an epitopeof a protein, that interacts specifically with a specific bindingmolecule. Antibodies, either member of a receptor/ligand pair, syntheticpolyamides (Dervan and Burli, Sequence-specific DNA recognition bypolyamides. Curr Opin Chem Biol, 3(6):688-93 (1999); Wemmer and Dervan,Targeting the minor groove of DNA. Curr Opin Struct Biol, 7(3):355-61(1997)), nucleic acid probes, and other molecules with specific bindingaffinities are examples of specific binding molecules, useful as theaffinity portion of a reporter binding molecule.

A specific binding molecule that interacts specifically with aparticular analyte is said to be specific for that analyte. For example,where the specific binding molecule is an antibody that associates witha particular antigen, the specific binding molecule is said to bespecific for that antigen. The antigen is the analyte. A reportermolecule containing the specific binding molecule can also be referredto as being specific for a particular analyte. Specific bindingmolecules preferably are antibodies, ligands, binding proteins, receptorproteins, haptens, aptamers, carbohydrates, synthetic polyamides,peptide nucleic acids, or oligonucleotides. Preferred binding proteinsare DNA binding proteins. Preferred DNA binding proteins are zinc fingermotifs, leucine zipper motifs, helix-turn-helix motifs. These motifs canbe combined in the same specific binding molecule.

Antibodies useful as the affinity portion of reporter binding agents,can be obtained commercially or produced using well established methods.For example, Johnstone and Thorpe, Immunochemistry In Practice(Blackwell Scientific Publications, Oxford, England, 1987) on pages30-85, describe general methods useful for producing both polyclonal andmonoclonal antibodies. The entire book describes many general techniquesand principles for the use of antibodies in assay systems.

Properties of zinc fingers, zinc finger motifs, and their interactions,are described by Nardelli et al., Zinc finger-DNA recognition: analysisof base specificity by site-directed mutagenesis. Nucleic Acids Res,20(16):4137-44 (1992), Jamieson et al., In vitro selection of zincfingers with altered DNA-binding specificity. Biochemistry,33(19):5689-95 (1994), Chandrasegaran and Smith, Chimeric restrictionenzymes: what is next? Biol Chem, 380(7-8):841-8 (1999), and Smith etal., A detailed study of the substrate specificity of a chimericrestriction enzyme. Nucleic Acids Res, 27(2):674-81 (1999).

One form of specific binding molecule is an oligonucleotide oroligonucleotide derivative. Such specific binding molecules are designedfor and used to detect specific nucleic acid sequences. Thus, theanalyte for oligonucleotide specific binding molecules are nucleic acidsequences. The analyte can be a nucleotide sequence within a largernucleic acid molecule. An oligonucleotide specific binding molecule canbe any length that supports specific and stable hybridization betweenthe reporter binding probe and the analyte. For this purpose, a lengthof 10 to 40 nucleotides is preferred, with an oligonucleotide specificbinding molecule 16 to 25 nucleotides long being most preferred. It ispreferred that the oligonucleotide specific binding molecule is peptidenucleic acid. Peptide nucleic acid forms a stable hybrid with DNA. Thisallows a peptide nucleic acid specific binding molecule to remain firmlyadhered to the target sequence during subsequent amplification anddetection operations.

This useful effect can also be obtained with oligonucleotide specificbinding molecules by making use of the triple helix chemical bondingtechnology described by Gasparro et al., Nucleic Acids Res.,22(14):2845-2852 (1994). Briefly, the oligonucleotide specific bindingmolecule is designed to form a triple helix when hybridized to a targetsequence. This is accomplished generally as known, preferably byselecting either a primarily homopurine or primarily homopyrimidinetarget sequence. The matching oligonucleotide sequence which constitutesthe specific binding molecule will be complementary to the selectedtarget sequence and thus be primarily homopyrimidine or primarilyhomopurine, respectively. The specific binding molecule (correspondingto the triple helix probe described by Gasparro et al.) contains achemically linked psoralen derivative. Upon hybridization of thespecific binding molecule to a target sequence, a triple helix forms. Byexposing the triple helix to low wavelength ultraviolet radiation, thepsoralen derivative mediates cross-linking of the probe to the targetsequence.

G. Reporter Signal Fusions

Reporter signal fusions are reporter signal peptides joined with aprotein or peptide of interest in a single amino acid segment (that is,a fusion protein). Such fusions of proteins and peptides of interestwith reporter signal peptides can be expressed as a fusion protein orpeptide from a nucleic acid molecule encoding the amino acid segmentthat constitutes the fusion. A reporter signal fusion nucleic acidmolecule or reporter signal nucleic acid segment refers to a nucleicacid molecule or nucleic acid sequence, respectively, that encodes areporter signal fusion. Although reference is made above and elsewhereherein to detection of, and fusion with, a “protein” or “proteins,” thedisclosed reporter signal fusions encompass fusions with proteins,peptides, and fragments of proteins or peptides. Thus, reference to aprotein herein is intended to refer to proteins, peptides, and fragmentsof proteins or peptides unless the context clearly indicates otherwise.

As used herein “reporter signal fusion” refers to a protein, peptide, orfragment of a protein or peptide to which a reporter signal peptide isfused (that is, joined by peptide bond(s) in the same polypeptide chain)unless the context clearly indicates otherwise. The reporter signalpeptide and the protein of interest involved in a reporter signal fusionneed not be directly fused. That is, other amino acids, amino acidsequences, and/or peptide elements can intervene. For example, anepitope tag, if present, can be located between the protein of interestand the reporter signal peptide in a reporter signal fusion. Thereporter signal peptide(s) can be fused to a protein in any arrangement,such as at the N-terminal end of the protein, at the C-terminal end ofthe protein, in or at domain junctions, or at any other appropriatelocation in the protein. In some forms of the method, it is desirablethat the protein remain functional. In such cases, terminal fusions orinter-domain fusions are preferably. Those of skill in the art ofprotein fusions generally know how to design fusions where the proteinof interest remains functional. In other embodiments, it is notnecessary that the protein remain functional in which case the reportersignal peptide and protein can have any desired structural organization.

A given reporter signal fusion can include one or more reporter signalpeptides and one or more proteins or peptides of interest. In addition,reporter signal fusions can include one or more amino acids, amino acidsequences, and/or peptide elements. The disclosed reporter signalfusions comprise a single, contiguous polypeptide chain. Thus, althoughmultiple amino acid segments can be part of the same contiguouspolypeptide chain, all of the components (that is, the reporter signalpeptide(s) and protein(s) and peptide(s) of interest) of a given aminoacid segment are part of the same contiguous polypeptide chain.

Reporter signal fusions can be produced by expression from nucleic acidmolecules encoding the fusions. Thus, the disclosed fusions generallycan be designed by designing nucleic acid segments that encode aminoacid segments where the amino acid segments comprise a reporter signalpeptide and a protein or peptide of interest. A given nucleic acidmolecule can comprise one or more nucleic acid segments. A given nucleicacid segment can encode one or more amino acid segments. A given aminoacid segment can include one or more reporter signal peptides and one ormore proteins or peptides of interest. The disclosed amino acid segmentsconsist of a single, contiguous polypeptide chain. Thus, althoughmultiple amino acid segments can be part of the same contiguouspolypeptide chain, all of the components (that is, the reporter signalpeptide(s) and protein(s) and peptide(s) of interest) of a given aminoacid segment are part of the same contiguous polypeptide chain.

Reporter signal fusions can include other components besides a proteinof interest and a reporter signal peptide. For example, reporter signalfusions can include epitope tags or flag peptides (see, for example,Groth et al. (2000) A phage integrase directs efficient site-specificintegration in human cells. Proc Natl Acad Sci USA 97:5995-6000).Epitope tags and flag peptides can serve as tags by which reportersignal fusions can be separated, distinguished, associated, and/orbound. The use of epitope tags and flag peptides generally is known andcan be adapted for use in the disclosed reporter signal fusions.

Reporter signal peptides can be fragmented, decomposed, reacted,derivatized, or otherwise modified, preferably in a characteristic way.This allows a protein to which the reporter signal peptide is fused tobe identified by detection of one or more of the products of thereporter signal fusion following fragmentation, decomposition, reaction,derivatization, or other modification of the reporter signal peptide.The protein can also be identified by the correlated detection of thereporter signal fusion and one or more of the products of the reportersignal fusion following fragmentation, decomposition, reaction,derivatization, or other modification of the reporter signal peptide.The alteration of the reporter signal peptide will alter the reportersignal fusion in a characteristic and detectable way. Together, thedetection of a characteristic reporter signal fusion and acharacteristic product of (that is, altered form of) the reporter signalfusion can uniquely identify the protein (although the altered formalone can be detected, if desired). In this way, expression of one ormore proteins can be detected, either alone or together (for example, ina multiplex assay). Further, expression of one or more proteins in oneor more samples can be detected in a multiplex manner. Preferably, formass spectrometry reporter signals, the reporter signal peptides arefragmented to yield fragments of similar charge but different mass.

Preferably, the reporter signal peptides are designed to be fragmentedto yield fragments of similar charge but different mass. This allowseach reporter signal fusion (and/or each reporter signal peptide) in aset to be distinguished by the different mass-to-charge ratios of thefragments of (that is, altered forms of) the reporter signal peptides.This is possible since the fragments of the different reporter signalpeptides (or the fragments of the reporter signal fusions) can bedesigned to have different mass-to-charge ratios. In the disclosedmethod, this allows each reporter signal fusion to be distinguished bythe mass-to-charge ratios of the reporter signal fusions afterfragmentation of the reporter signal peptide.

Alteration of reporter signals peptides in reporter signal fusions canproduce a variety of altered compositions. Any or all of these alteredforms can be detected. For example, the altered form of the reportersignal peptide can be detected, the altered form of the amino acidsegment (which contains the reporter signal peptide) can be detected,the altered form of a subsegment of the amino acid segment can bedetected, or a combination of these can be detected. Where the reportersignal peptide is altered by fragmentation, the result generally will bea fragment of the reporter signal peptide and an altered form of theamino acid segment containing the protein or peptide of interest and aportion of the reporter signal peptide (that is, the portion not in thereporter signal peptide fragment).

The protein or peptide of interest also can be fragmented. The resultwould be a subsegment of the amino acid segment. The amino acidsubsegment would contain the reporter signal peptide and a portion ofthe protein or peptide of interest. When the reporter signal peptide inan amino acid subsegment is altered (which can occur before, during, orafter fragmentation of the amino acid segment), the result is an alteredform of the amino acid subsegment (and an altered form of the reportersignal peptide). This altered form of amino acid subsegment can bedetected. Where the reporter signal peptide is altered by fragmentation,the result generally will be a fragment of the reporter signal peptideand an altered form of (that is, fragment of) the amino acid subsegment.In this case, the altered form of the amino acid subsegment, which isalso referred to herein as a reporter signal fusion fragment, willcontain a portion of the protein or peptide of interest and a portion ofthe reporter signal peptide (that is, the portion not in the reportersignal peptide fragment).

As with reporter signals generally, reporter signal fusions (alsoreferred to as amino acid segments), reporter signal fusion fragments(also referred to as subsegments of the reporter signal fusions), orreporter signal peptides can be used in sets where the reporter signalfusions, reporter signal fusion fragments, or reporter signal peptidesin a set can have one or more common properties that allow the reportersignal fusions, reporter signal fusion fragments, or reporter signalpeptides to be separated or distinguished from molecules lacking thecommon property. In the case of reporter signal fusions, amino acidsegments and amino acid subsegments can be used in sets where the aminoacid segments and amino acid subsegments in a set can have one or morecommon properties that allow the amino acid segments and amino acidsubsegments, respectively, to be separated or distinguished frommolecules lacking the common property. In general, the component(s) ofthe reporter signal fusions having common properties can depend on thecomponent(s) to be detected and/or the mode of the method being used.

A variety of different properties can be used as the common physicalproperty used to separate reporter signal fusions, reporter signalfusion fragments, and/or reporter signal peptides from other moleculeslacking the common property. For example, physical properties useful ascommon properties include mass-to-charge ratio, mass, charge,isoelectric point, hydrophobicity, chromatography characteristics, anddensity. It is preferred that the physical property shared by reportersignal fusions, reporter signal fusion fragments, or reporter signalpeptides in a set (and used to distinguish or separate the reportersignal fusions, reporter signal fusion fragments, or reporter signalpeptides from other molecules) is an overall property of the reportersignal fusions, reporter signal fusion fragments, or reporter signalpeptides (for example, overall mass, overall charge, isoelectric point,overall hydrophobicity, etc.) rather than the mere presence of a featureor moiety (for example, an affinity tag, such as biotin). Suchproperties are referred to herein as “overall” properties (and thus,reporter signal fusions, reporter signal fusion fragments, or reportersignal peptides in a set would be referred to as sharing a “commonoverall property”). It should be understood that reporter signalfusions, reporter signal fusion fragments, or reporter signal peptidescan have features and moieties, such as affinity tags, and that suchfeatures and moieties can contribute to the common overall property (bycontributing mass, for example). However, such limited and isolatedfeatures and moieties would not serve as the sole basis of the commonoverall property.

It is preferred that the common property of reporter signal fusions,reporter signal fusion fragments, or reporter signal peptides is not anaffinity tag. Nevertheless, even in such a case, reporter signalfusions, reporter signal fusion fragments, or reporter signal peptidesthat otherwise have a common property may also include an affinitytag—and in fact may all share the same affinity tag—so long as anothercommon property is present that can be (and, in some embodiments of thedisclosed method, is) used to separate reporter signal fusions, reportersignal fusion fragments, or reporter signal peptides sharing the commonproperty from other molecules lacking the common property. With this inmind, it is preferred that, if chromatography or other separationtechniques are used to separate reporter signal fusions, reporter signalfusion fragments, or reporter signal peptides based on the commonproperty, the affinity be based on an overall physical property of thereporter signal fusions, reporter signal fusion fragments, or reportersignal peptides and not on the presence of, for example, a feature ormoiety such as an affinity tag. As used herein, a common property is aproperty shared by a set of components (such as reporter signal fusions,reporter signal fusion fragments, or reporter signal peptides). That is,the components have the property “in common.” It should be understoodthat reporter signal fusions, reporter signal fusion fragments, orreporter signal peptides in a set may have numerous properties incommon. However, as used herein, the common properties of reportersignal fusions, reporter signal fusion fragments, or reporter signalpeptides referred to are only those used in the disclosed method todistinguish and/or separate the reporter signal fusions, reporter signalfusion fragments, or reporter signal peptides sharing the commonproperty from molecules that lack the common property.

In preferred embodiments, reporter signal peptides, reporter signalfusions (or amino acid segments), nucleic acid segments encodingreporter signal fusions, and/or nucleic acid molecules comprisingnucleic acid segments encoding reporter signal fusions are used in setswhere the reporter signal peptides, the reporter signal fusions, and/orsubsegments of the reporter signal fusions constituting or present inthe set have similar properties (such as similar mass-to-charge ratios).The similar properties allow the reporter signals, the reporter signalfusions, or subsegments of the reporter signal fusions to bedistinguished and/or separated from other molecules lacking one or moreof the properties. Preferably, the reporter signals, the reporter signalfusions, or subsegments of the reporter signal fusions constituting orpresent in a set have the same mass-to-charge ratio (m/z). That is, thereporter signals, the reporter signal fusions, or subsegments of thereporter signal fusions in a set are isobaric. This allows the reportersignals, the reporter signal fusions, or subsegments of the reportersignal fusions to be separated precisely from other molecules based onmass-to-charge ratio. The result of the filtering is a huge increase inthe signal to noise ratio (S/N) for the system, allowing more sensitiveand accurate detection.

Sets of reporter signal fusions (also referred to as amino acidsegments), reporter signal fusion fragments (also referred to assubsegments of the reporter signal fusions or amino acid subsegments),reporter signal peptides, nucleic acid segments encoding reporter signalfusions, or nucleic acid molecules comprising nucleic acid segmentsencoding reporter signal fusions can have any number of reporter signalfusions, reporter signal fusion fragments, reporter signal peptides,nucleic acid segments encoding reporter signal fusions, or nucleic acidmolecules comprising nucleic acid segments encoding reporter signalfusions. For example, sets of reporter signal fusions, reporter signalfusion fragments, reporter signal peptides, nucleic acid segmentsencoding reporter signal fusions, or nucleic acid molecules comprisingnucleic acid segments encoding reporter signal fusions can have one, twoor more, three or more, four or more, five or more, six or more, sevenor more, eight or more, nine or more, ten or more, twenty or more,thirty or more, forty or more, fifty or more, sixty or more, seventy ormore, eighty or more, ninety or more, one hundred or more, two hundredor more, three hundred or more, four hundred or more, or five hundred ormore different reporter signal fusions, reporter signal fusionfragments, reporter signal peptides, nucleic acid segments encodingreporter signal fusions, or nucleic acid molecules comprising nucleicacid segments encoding reporter signal fusions. Although specificnumbers of reporter signal fusions, reporter signal fusion fragments,reporter signal peptides, nucleic acid segments encoding reporter signalfusions, and nucleic acid molecules comprising nucleic acid segmentsencoding reporter signal fusions, and specific endpoints for ranges ofthe number of reporter signal fusions, reporter signal fusion fragments,reporter signal peptides, nucleic acid segments encoding reporter signalfusions, and nucleic acid molecules comprising nucleic acid segmentsencoding reporter signal fusions, are recited, each and every specificnumber of reporter signal fusions, reporter signal fusion fragments,reporter signal peptides, nucleic acid segments encoding reporter signalfusions, and nucleic acid molecules comprising nucleic acid segmentsencoding reporter signal fusions, and each and every specific endpointof ranges of numbers of reporter signal fusions, reporter signal fusionfragments, reporter signal peptides, nucleic acid segments encodingreporter signal fusions, and nucleic acid molecules comprising nucleicacid segments encoding reporter signal fusions, are specificallycontemplated, although not explicitly listed, and each and everyspecific number of reporter signal fusions, reporter signal fusionfragments, reporter signal peptides, nucleic acid segments encodingreporter signal fusions, and nucleic acid molecules comprising nucleicacid segments encoding reporter signal fusions, and each and everyspecific endpoint of ranges of numbers of reporter signal fusions,reporter signal fusion fragments, reporter signal peptides, nucleic acidsegments encoding reporter signal fusions, and nucleic acid moleculescomprising nucleic acid segments encoding reporter signal fusions, arehereby specifically described.

The reporter signal fusions are preferably detected using massspectrometry which allows sensitive distinctions between molecules basedon their mass-to-charge ratios. A set of isobaric reporter signalpeptides or reporter signal fusions can be used for multiplex labelingand/or detection of the expression of many genes, proteins, vectors,expression constructs, cells, cell lines, and organisms since thereporter signal peptide fragments can be designed to have a large rangeof masses, with each mass individually distinguishable upon detection.Where the same gene, protein, vectors, expression construct, cell, cellline, or organism (or the same type of gene, protein, vector, expressionconstruct, cell, cell line, or organism) is labeled with a set ofreporter signal fusions that are isobaric or that include isobaricreporter signal peptides (by, for example, “labeling” the same gene,protein, vector, expression construct, cell, cell line, or organism indifferent samples), the set of reporter signal fusions or reportersignal peptides that results will also be isobaric. Fragmentation of thereporter signal peptides will split the set of reporter signal peptidesinto individually detectable reporter signal fusion fragments andreporter signal peptide fragments of characteristically different mass.

Reporter signal fusions can be expressed in any suitable manner. Forexample, nucleic acid sequences and nucleic acid segments encodingreporter signal fusions can be expressed in vitro, in cells, and/or incells in organism. Many techniques and systems for expression of nucleicacid sequences and proteins are known and can be used with the disclosedreporter signal fusions. For example, many expression sequences, vectorsystems, transformation and transfection techniques, and transgenicorganism production methods are known and can be used with the disclosedreporter signal peptide method and compositions. Systems are known forintegration of nucleic acid constructs into chromosomes of cells andorganisms (see, for example, Groth et al. (2000) A phage integrasedirects efficient site-specific integration in human cells. Proc NatlAcad Sci USA 97:5995-6000; Hong et al. (2001) Development of twobacterial artificial chromosome shuttle vectors for arecombination-based cloning and regulated expression of large genes inmammalian cells. Analytical Biochemistry 291:142-148) which can be usedwith the disclosed nucleic acid molecules and segments encoding reportersignal fusions or to form nucleic acid segment encoding reporter signalfusions.

As used herein, an expression sample is a sample that contains, or mightcontain, one or more reporter signal fusions expressed from a nucleicacid molecule. An expression sample to be analyzed can be subjected tofractionation or separation to reduce the complexity of the samples.Fragmentation and fractionation can also be used together in the sameassay. Such fragmentation and fractionation can simplify and extend theanalysis of the expression.

Nucleic acid molecules encoding reporter signal fusions can be used insets where the reporter signal peptides in the reporter signal fusionsencoded by a set of nucleic acid molecules can have one or more commonproperties that allow the reporter signal peptides to be separated ordistinguished from molecules lacking the common property. Similarly,nucleic acid molecules encoding amino acid segments can be used in setswhere the reporter signal peptides in the amino acid segments encoded bya set of nucleic acid molecules can have one or more common propertiesthat allow the reporter signal peptides to be separated or distinguishedfrom molecules lacking the common property. Nucleic acid moleculesencoding amino acid segments can be used in sets where the amino acidsegments encoded by a set of nucleic acid molecules can have one or morecommon properties that allow the amino acid segments to be separated ordistinguished from molecules lacking the common property.

Nucleic acid segments (which, generally, are part of nucleic acidmolecules) encoding reporter signal fusions can be used in sets wherethe reporter signal peptides in the reporter signal fusions encoded by aset of nucleic acid segments can have one or more common properties thatallow the reporter signal peptides to be separated or distinguished frommolecules lacking the common property. Similarly, nucleic acid segmentsencoding amino acid segments can be used in sets where the reportersignal peptides in the amino acid segments encoded by a set of nucleicacid molecules can have one or more common properties that allow thereporter signal peptides to be separated or distinguished from moleculeslacking the common property. Nucleic acid segments encoding amino acidsegments can be used in sets where the amino acid segments encoded by aset of nucleic acid molecules can have one or more common propertiesthat allow the amino acid segments to be separated or distinguished frommolecules lacking the common property. Other relationships betweenmembers of the sets of nucleic acid molecules, nucleic acid segments,amino acid segments, reporter signal peptides, and proteins of interestare contemplated.

Reporter signal fusions allow sensitive and multiplex detection ofexpression of particular proteins and peptides of interest, and/or ofthe genes, vectors, and expression constructs encoding the proteins andpeptides of interest. The disclosed reporter signal fusions can also beused for any purpose including as a source of reporter signals for otherforms of the disclosed method and compositions.

H. Reporter Signal/Analyte Conjugates

Compositions where reporter signals are associated with, incorporatedinto, or otherwise linked to the analytes are referred to as reportersignal/analyte conjugates. Such conjugates include reporter signalsassociated with analytes, such as a reporter signal probe hybridized toa nucleic acid sequence; reporter signals covalently coupled toanalytes, such as reporter signals linked to proteins via a linkinggroup; and reporter signals incorporated into analytes, such as fusionsbetween a protein of interest and a peptide reporter signal.

Reporter signal/analyte conjugates can be altered, generally throughalteration of the reporter signal portion of the conjugate, such thatthe altered forms of different reporter signals, altered forms ofdifferent reporter signal/analyte conjugates, or both, can bedistinguished from each other. Where the reporter signal or reportersignal/analyte conjugate is altered by fragmentation, any, some, or allof the fragments can be distinguished from each other, depending on theembodiment. For example, where reporter signal/analyte conjugates arefragmented into two parts (with the break point in the reporter signalportion), either the reporter signal fragment, the reportersignal/analyte fragment, or both can be distinguished.

Sets of reporter signal/analyte conjugates can be used where two or moreof the reporter signal/analyte conjugates in a set have one or morecommon properties that allow the reporter signal/analyte conjugateshaving the common property to be distinguished and/or separated fromother molecules lacking the common property. In still other embodiments,analytes can be fragmented (prior to or following conjugation) toproduce reporter signal/analyte fragment conjugates (which can bereferred to as fragment conjugates). In such cases, sets of fragmentconjugates can be used where two or more of the fragment conjugates in aset have one or more common properties that allow the fragmentconjugates having the common property to be distinguished and/orseparated from other molecules lacking the common property. It should beunderstood that fragmented analytes can be considered analytes in theirown right. In this light, reference to fragmented analytes is made forconvenience and clarity in describing certain embodiments and to allowreference to both the base analyte and the fragmented analyte.

Sets of reporter signal/analyte conjugates or reporter signal/analytefragment conjugates (fragment conjugates) can have any number ofreporter signal/analyte conjugates or reporter signal/analyte fragmentconjugates. For example, sets of reporter signal/analyte conjugates orreporter signal/analyte fragment conjugates can have one, two or more,three or more, four or more, five or more, six or more, seven or more,eight or more, nine or more, ten or more, twenty or more, thirty ormore, forty or more, fifty or more, sixty or more, seventy or more,eighty or more, ninety or more, one hundred or more, two hundred ormore, three hundred or more, four hundred or more, or five hundred ormore different reporter signal/analyte conjugates or reportersignal/analyte fragment conjugates. Although specific numbers ofreporter signal/analyte conjugates and reporter signal/analyte fragmentconjugates, and specific endpoints for ranges of the number of reportersignal/analyte conjugates and reporter signal/analyte fragmentconjugates, are recited, each and every specific number of reportersignal/analyte conjugates and reporter signal/analyte fragmentconjugates, and each and every specific endpoint of ranges of numbers ofreporter signal/analyte conjugates and reporter signal/analyte fragmentconjugates, are specifically contemplated, although not explicitlylisted, and each and every specific number of reporter signal/analyteconjugates and reporter signal/analyte fragment conjugates, and each andevery specific endpoint of ranges of numbers of reporter signal/analyteconjugates and reporter signal/analyte fragment conjugates, are herebyspecifically described.

As indicated above, reporter signals conjugated with analytes can bealtered while in the conjugate and distinguished. Conjugated reportersignals can also be dissociated or separated, in whole or in part, fromthe conjugated analytes prior to their alteration. Where the reportersignals are dissociated (in whole or in part) from the analytes, themethod can be performed such that the fact of association between theanalyte and reporter signal is part of the information obtained when thereporter signal is detected. In other words, the fact that the reportersignal may be dissociated from the analyte for detection does notobscure the information that the detected reporter signal was associatedwith the analyte.

As used herein, reporter signal conjugate refers both to reportersignal/analyte conjugates and to other components of the disclosedmethod such as reporter molecules.

As with reporter signals generally, reporter signal/analyte conjugatesand reporter signal/analyte fragment conjugates can be used in setswhere the reporter signal/analyte conjugates or fragment conjugates in aset can have one or more common properties that allow the reportersignal/analyte conjugates or fragment conjugates to be separated ordistinguished from molecules lacking the common property. As withreporter signals generally, a variety of different properties can beused as the common physical property used to separate reportersignal/analyte conjugates or fragment conjugates from other moleculeslacking the common property. For example, physical properties useful ascommon properties include mass-to-charge ratio, mass, charge,isoelectric point, hydrophobicity, chromatography characteristics, anddensity. It is preferred that the physical property shared by reportersignal/analyte conjugates or fragment conjugates in a set (and used todistinguish or separate the reporter signal/analyte conjugates orfragment conjugates from other molecules) is an overall property of thereporter signal/analyte conjugates or fragment conjugates (for example,overall mass, overall charge, isoelectric point, overall hydrophobicity,etc.) rather than the mere presence of a feature or moiety (for example,an affinity tag, such as biotin). Such properties are referred to hereinas “overall” properties (and thus, reporter signal/analyte conjugates orfragment conjugates in a set would be referred to as sharing a “commonoverall property”). It should be understood that reporter signal/analyteconjugates or fragment conjugates can have features and moieties, suchas affinity tags, and that such features and moieties can contribute tothe common overall property (by contributing mass, for example).However, such limited and isolated features and moieties would not serveas the sole basis of the common overall property.

It is preferred that the common property of reporter signal/analyteconjugates or fragment conjugates is not an affinity tag. Nevertheless,even in such a case, reporter signal/analyte conjugates or fragmentconjugates that otherwise have a common property may also include anaffinity tag—and in fact may all share the same affinity tag—so long asanother common property is present that can be (and, in some embodimentsof the disclosed method, is) used to separate reporter signal/analyteconjugates or fragment conjugates sharing the common property from othermolecules lacking the common property. With this in mind, it ispreferred that, if chromatography or other separation techniques areused to separate reporter signal/analyte conjugates or fragmentconjugates based on the common property, the affinity be based on anoverall physical property of the reporter signal/analyte conjugates orfragment conjugates and not on the presence of, for example, a featureor moiety such as an affinity tag. As used herein, a common property isa property shared by a set of components (such as reportersignal/analyte conjugates or fragment conjugates). That is, thecomponents have the property “in common.” It should be understood thatreporter signal/analyte conjugates or fragment conjugates in a set mayhave numerous properties in common. However, as used herein, the commonproperties of reporter signal/analyte conjugates or fragment conjugatesreferred to are only those used in the disclosed method to distinguishand/or separate the reporter signal/analyte conjugates or fragmentconjugates sharing the common property from molecules that lack thecommon property.

I. Capture Arrays

A capture array (also referred to herein as an array) includes aplurality of capture tags immobilized on a solid-state substrate,preferably at identified or predetermined locations on the solid-statesubstrate. In this context, plurality of capture tags refers to amultiple capture tags each having a different structure. Preferably,each predetermined location on the array (referred to herein as an arrayelement) has one type of capture tag (that is, all the capture tags atthat location have the same structure). Each location will have multiplecopies of the capture tag. The spatial separation of capture tags ofdifferent structure in the array allows separate detection andidentification of analytes that become associated with the capture tags.If a decoding tag is detected at a given location in a capture array, itindicates that the analyte corresponding to that array element waspresent in the target sample.

Solid-state substrates for use in capture arrays can include any solidmaterial to which capture tags can be coupled, directly or indirectly.This includes materials such as acrylamide, cellulose, nitrocellulose,glass, polystyrene, polyethylene vinyl acetate, polypropylene,polymethacrylate, polyethylene, polyethylene oxide, glass,polysilicates, polycarbonates, teflon, fluorocarbons, nylon, siliconrubber, polyanhydrides, polyglycolic acid, polylactic acid,polyorthoesters, polypropylfumerate, collagen, glycosaminoglycans, andpolyamino acids. Solid-state substrates can have any useful formincluding thin films or membranes, beads, bottles, dishes, disks,compact disks, fibers, optical fibers, woven fibers, shaped polymers,particles and microparticles. A preferred form for a solid-statesubstrate is a compact disk.

Although preferred, it is not required that a given capture array be asingle unit or structure. The set of capture tags may be distributedover any number of solid supports. For example, at one extreme, eachcapture tag may be immobilized in a separate reaction tube or container.Arrays may be constructed upon non permeable or permeable supports of awide variety of support compositions such as those described above. Thearray spot sizes and density of spot packing vary over a tremendousrange depending upon the process(es) and material(s) used.

Methods for immobilizing antibodies and other proteins to substrates arewell established. Immobilization can be accomplished by attachment, forexample, to aminated surfaces, carboxylated surfaces or hydroxylatedsurfaces using standard immobilization chemistries. Examples ofattachment agents are cyanogen bromide, succinimide, aldehydes, tosylchloride, avidin-biotin, photocrosslinkable agents, epoxides andmaleimides. A preferred attachment agent is glutaraldehyde. These andother attachment agents, as well as methods for their use in attachment,are described in Protein immobilization: fundamentals and applications,Richard F. Taylor, ed. (M. Dekker, New York, 1991), Johnstone andThorpe, Immunochemistry In Practice (Blackwell Scientific Publications,Oxford, England, 1987) pages 209-216 and 241-242, and ImmobilizedAffinity Ligands, Craig T. Hermanson et al., eds. (Academic Press, NewYork, 1992). Antibodies can be attached to a substrate by chemicallycross-linking a free amino group on the antibody to reactive side groupspresent within the substrate. For example, antibodies may be chemicallycross-linked to a substrate that contains free amino or carboxyl groupsusing glutaraldehyde or carbodiimides as cross-linker agents. In thismethod, aqueous solutions containing free antibodies are incubated withthe solid-state substrate in the presence of glutaraldehyde orcarbodiimide. For crosslinking with glutaraldehyde the reactants can beincubated with 2% glutaraldehyde by volume in a buffered solution suchas 0.1 M sodium cacodylate at pH 7.4. Other standard immobilizationchemistries are known by those of skill in the art.

Methods for immobilization of oligonucleotides to solid-state substratesare well established. Oligonucleotide capture tags can be coupled tosubstrates using established coupling methods. For example, suitableattachment methods are described by Pease et al., Proc. Natl. Acad. Sci.USA 91(11):5022-5026 (1994), Khrapko et al., Mol Biol (Mosk) (USSR)25:718-730 (1991), U.S. Pat. No. 5,871,928 to Fodor et al., U.S. Pat.No. 5,654,413 to Brenner, U.S. Pat. No. 5,429,807, and U.S. Pat. No.5,599,695 to Pease et al. A method for immobilization of 3′-amineoligonucleotides on casein-coated slides is described by Stimpson etal., Proc. Natl. Acad. Sci. USA 92:6379-6383 (1995). A preferred methodof attaching oligonucleotides to solid-state substrates is described byGuo et al., Nucleic Acids Res. 22:5456-5465 (1994).

Planar array technology has been utilized for many years (Shalon, D., S.J. Smith, and P. O. Brown, A DNA microarray system for analyzing complexDNA samples using two-colorfluorescent probe hybridization. Genome Res,1996. 6(7): p. 639-45, Singh-Gasson, S., et al., Maskless fabrication oflight-directed oligonucleotide microarrays using a digital micromirrorarray. Nat Biotechnol, 1999. 17(10): p. 974-8, Southern, E. M., U.Maskos, and J. K. Elder, Analyzing and comparing nucleic acid sequencesby hybridization to arrays of oligonucleotides: evaluation usingexperimental models. Genomics, 1992. 13(4): p. 1008-17, Nizetic, D., etal., Construction, arraying, and high-density screening of large insertlibraries of human chromosomes X and 21: their potential use asreference libraries. Proc Natl Acad Sci USA, 1991. 88(8): p. 3233-7, VanOss, C. J., R. J. Good, and M. K. Chaudhury, Mechanism of DNA (Southern)and protein (Western) blotting on cellulose nitrate and other membranes.J Chromatogr, 1987. 391(1): p. 53-65, Ramsay, G., DNA chips:state-of-the art. Nat Biotechnol, 1998. 16(1): p. 40-4, Schena, M., etal., Parallel human genome analysis: microarray-based expressionmonitoring of 1000 genes. Proc Natl Acad Sci USA, 1996. 93(20): p.10614-9, Lipshutz, R. J., et al., High density synthetic oligonucleotidearrays. Nat Genet, 1999. 21(1 Suppl): p. 20-4, Pease, A. C., et al.,Light-generated oligonucleotide arrays for rapid DNA sequence analysis.Proc Natl Acad Sci USA, 1994. 91(11): p. 5022-6, Maier, E., et al.,Application of robotic technology to automated sequence fingerprintanalysis by oligonucleotide hybridisation. J Biotechnol, 1994. 35(2-3):p. 191-203, Vasiliskov, A. V., et al., Fabrication of microarray ofgel-immobilized compounds on a chip by copolymerization. Biotechniques,1999. 27(3): p. 592-4, 596-8, 600 passim, and Yershov, G., et al., DNAanalysis and diagnostics on oligonucleotide microchips. Proc Natl AcadSci USA, 1996. 93(10): p. 4913-8).

Oligonucleotide capture tags in arrays can also be designed to havesimilar hybrid stability. This would make hybridization of fragments tosuch capture tags more efficient and reduce the incidence of mismatchhybridization. The hybrid stability of oligonucleotide capture tags canbe calculated using known formulas and principles of thermodynamics(see, for example, Santa Lucia et al., Biochemistry 35:3555-3562 (1996);Freier et al., Proc. Natl. Acad. Sci. USA 83:9373-9377 (1986); Breslaueret al., Proc. Natl. Acad. Sci. USA 83:3746-3750 (1986)). The hybridstability of the oligonucleotide capture tags can be made more similar(a process that can be referred to as smoothing the hybrid stabilities)by, for example, chemically modifying the capture tags (Nguyen et al.,Nucleic Acids Res. 25(15):3059-3065 (1997); Hohsisel, Nucleic Acids Res.24(3):430-432 (1996)). Hybrid stability can also be smoothed by carryingout the hybridization under specialized conditions (Nguyen et al.,Nucleic Acids Res. 27(6):1492-1498 (1999); Wood et al., Proc. Natl.Acad. Sci. USA 82(6):1585-1588 (1985)).

Another means of smoothing hybrid stability of the oligonucleotidecapture tags is to vary the length of the capture tags. This would allowadjustment of the hybrid stability of each capture tag so that all ofthe capture tags had similar hybrid stabilities (to the extentpossible). Since the addition or deletion of a single nucleotide from acapture tag will change the hybrid stability of the capture tag by afixed increment, it is understood that the hybrid stabilities of thecapture tags in a capture array will not be equal. For this reason,similarity of hybrid stability as used herein refers to any increase inthe similarity of the hybrid stabilities of the capture tags (or, putanother way, any reduction in the differences in hybrid stabilities ofthe capture tags).

The efficiency of hybridization and ligation of oligonucleotide capturetags to sample fragments can also be improved by grouping capture tagsof similar hybrid stability in sections or segments of a capture arraythat can be subjected to different hybridization conditions. In thisway, the hybridization conditions can be optimized for particularclasses of capture tags.

J. Capture Tags

A capture tag is any compound that can be used to capture or separatecompounds or complexes having the capture tag. Preferably, a capture tagis a compound that interacts specifically with a particular molecule ormoiety. Preferably, the molecule or moiety that interacts specificallywith a capture tag is an analyte. It is to be understood that the termanalyte refers to both separate molecules and to portions of suchmolecules, such as an epitope of a protein, that interacts specificallywith a capture tag. Antibodies, either member of a receptor/ligand pair,synthetic polyamides (Dervan and Burli, Sequence-specific DNArecognition by polyamides. Curr Opin Chem Biol, 3(6):688-93 (1999);Wemmer and Dervan, Targeting the minor groove of DNA. Curr Opin StructBiol, 7(3):355-61 (1997)), nucleic acid probes, and other molecules withspecific binding affinities are examples of capture tags.

A capture tag that interacts specifically with a particular analyte issaid to be specific for that analyte. For example, where the capture tagis an antibody that associates with a particular antigen, the capturetag is said to be specific for that antigen. The antigen is the analyte.Capture tags preferably are antibodies, ligands, binding proteins,receptor proteins, haptens, aptamers, carbohydrates, syntheticpolyamides, peptide nucleic acids, or oligonucleotides. Preferredbinding proteins are DNA binding proteins. Preferred DNA bindingproteins are zinc finger motifs, leucine zipper motifs, helix-turn-helixmotifs. These motifs can be combined in the same capture tag.

Antibodies useful as the affinity portion of reporter binding agents,can be obtained commercially or produced using well established methods.For example, Johnstone and Thorpe, Immunochemistry In Practice(Blackwell Scientific Publications, Oxford, England, 1987) on pages30-85, describe general methods useful for producing both polyclonal andmonoclonal antibodies. The entire book describes many general techniquesand principles for the use of antibodies in assay systems.

Properties of zinc fingers, zinc finger motifs, and their interactions,are described by Nardelli et al., Zinc finger-DNA recognition: analysisof base specificity by site-directed mutagenesis. Nucleic Acids Res,20(16):4137-44 (1992), Jamieson et al., In vitro selection of zincfingers with altered DNA-binding specificity. Biochemistry,33(19):5689-95 (1994), Chandrasegaran and Smith, Chimeric restrictionenzymes: what is next? Biol Chem, 380(7-8):841-8 (1999), and Smith etal., A detailed study of the substrate specificity of a chimericrestriction enzyme. Nucleic Acids Res, 27(2):674-81 (1999).

One form of capture tag is an oligonucleotide or oligonucleotidederivative. Such capture tags are designed for and used to detectspecific nucleic acid sequences. Thus, the analyte for oligonucleotidecapture tags are nucleic acid sequences. The analyte can be a nucleotidesequence within a larger nucleic acid molecule. An oligonucleotidecapture tag can be any length that supports specific and stablehybridization between the capture tag and the analyte. For this purpose,a length of 10 to 40 nucleotides is preferred, with an oligonucleotidecapture tag 16 to 25 nucleotides long being most preferred. It ispreferred that the oligonucleotide capture tag is peptide nucleic acid.Peptide nucleic acid forms a stable hybrid with DNA. This allows apeptide nucleic acid capture tag to remain firmly adhered to the targetsequence during subsequent amplification and detection operations.

This useful effect can also be obtained with oligonucleotide capturetags by making use of the triple helix chemical bonding technologydescribed by Gasparro et al., Nucleic Acids Res., 22(14):2845-2852(1994). Briefly, the oligonucleotide capture tag is designed to form atriple helix when hybridized to a target sequence. This is accomplishedgenerally as known, preferably by selecting either a primarilyhomopurine or primarily homopyrimidine target sequence. The matchingoligonucleotide sequence which constitutes the capture tag will becomplementary to the selected target sequence and thus be primarilyhomopyrimidine or primarily homopurine, respectively. The capture tag(corresponding to the triple helix probe described by Gasparro et al.)contains a chemically linked psoralen derivative. Upon hybridization ofthe capture tag to a target sequence, a triple helix forms. By exposingthe triple helix to low wavelength ultraviolet radiation, the psoralenderivative mediates cross-linking of the probe to the target sequence.

K. Sample Arrays

A sample array includes a plurality of samples (for example, expressionsamples, tissue samples, protein samples) immobilized on a solid-statesubstrate, preferably at identified or predetermined locations on thesolid-state substrate. Preferably, each predetermined location on thesample array (referred to herein as an sample array element) has onetype of sample. The spatial separation of different samples in thesample array allows separate detection and identification of reportersignals (or reporter molecules or coding tags) that become associatedwith the samples. If a reporter signal is detected at a given locationin a sample array, it indicates that the analyte corresponding to thatreporter signal was present in the sample corresponding to that samplearray element.

Solid-state substrates for use in sample arrays can include any solidmaterial to which samples can be adhered, directly or indirectly. Thisincludes materials such as acrylamide, cellulose, nitrocellulose, glass,polystyrene, polyethylene vinyl acetate, polypropylene,polymethacrylate, polyethylene, polyethylene oxide, glass,polysilicates, polycarbonates, teflon, fluorocarbons, nylon, siliconrubber, polyanhydrides, polyglycolic acid, polylactic acid,polyorthoesters, polypropylfumerate, collagen, glycosaminoglycans, andpolyamino acids. Solid-state substrates can have any useful formincluding thin films or membranes, beads, bottles, dishes, disks,compact disks, fibers, optical fibers, woven fibers, shaped polymers,particles and microparticles. A preferred form for a solid-statesubstrate is a compact disk.

Although preferred, it is not required that a given sample array be asingle unit or structure. The set of samples may be distributed over anynumber of solid supports. For example, at one extreme, each sample maybe immobilized in a separate reaction tube or container. Sample arraysmay be constructed upon non permeable or permeable supports of a widevariety of support compositions such as those described above. The arrayspot sizes and density of spot packing vary over a tremendous rangedepending upon the process(es) and material(s) used. Methods foradhering or immobilizing samples and sample components to substrates arewell established.

A preferred form of sample array is a tissue arrays, where there aresmall tissue samples on a substrate. Such tissue microarrays exist, andare used, for example, in a cohort to study breast cancer. The disclosedmethod can be used, for example, to probe multiple analytes in multiplesamples. Sample arrays can be, for example, labeled with differentreporter signals, the whole support then introduced into source regionof a mass spec, and sampled by MALDI.

L. Decoding Tags

Decoding tags are any molecule or moiety that can be associated withcoding tags, directly or indirectly. Decoding tags are associated withreporter signals (making up a reporter molecule) to allow indirectassociation of the reporter signals with an analyte. Decoding tagspreferably are oligonucleotides, carbohydrates, synthetic polyamides,peptide nucleic acids, antibodies, ligands, proteins, haptens, zincfingers, aptamers, or mass labels.

Preferred decoding tags are molecules capable of hybridizingspecifically to an oligonucleotide coding tag. Most preferred arepeptide nucleic acid decoding tags. Oligonucleotide or peptide nucleicacid decoding tags can have any arbitrary sequence. The only requirementis hybridization to coding tags. The decoding tags can each be anylength that supports specific and stable hybridization between thecoding tags and the decoding tags. For this purpose, a length of 10 to35 nucleotides is preferred, with a decoding tag 15 to 20 nucleotideslong being most preferred.

Reporter molecules containing decoding tags preferably are capable ofbeing released by matrix-assisted laser desorption-ionization (MALDI) inorder to be separated and identified by time-of-flight (TOF) massspectroscopy, or by another detection technique. A decoding tag may beany oligomeric molecule that can hybridize to a coding tag. For example,a decoding tag can be a DNA oligonucleotide, an RNA oligonucleotide, ora peptide nucleic acid (PNA) molecule. Preferred decoding tags are PNAmolecules.

M. Coding Tags

Coding tags are molecules or moieties with which decoding tags canassociate. Coding tags can be any type of molecule or moiety that canserve as a target for decoding tag association. Preferred coding tagsare oligomers, oligonucleotides, or nucleic acid sequences. Coding tagscan also be a member of a binding pair, such as streptavidin or biotin,where its cognate decoding tag is the other member of the binding pair.Coding tags can also be designed to associate directly with some typesof reporter signals. For example, oligonucleotide coding tags can bedesigned to interact directly with peptide nucleic acid reporter signals(which are reporter signals composed of peptide nucleic acid).

The oligomeric base sequences of oligomeric coding tags can include RNA,DNA, modified RNA or DNA, modified backbone nucleotide-like oligomerssuch as peptide nucleic acid, methylphosphonate DNA, and 2′-O-methyl RNAor DNA. Oligomeric or oligonucleotide coding tags can have any arbitrarysequence. The only requirement is association with decoding tags(preferably by hybridization). In the disclosed method, multiple codingtags can become associated with a single analyte. The context of thesemultiple coding tags depends upon the technique used for signalamplification. Thus, where branched DNA is used, the branched DNAmolecule includes the multiple coding tags on the branches. Whereoligonucleotide dendrimers are used, the coding tags are on thedendrimer arms. Where rolling circle replication is used, multiplecoding tags result from the tandem repeats of complement of theamplification target circle sequence (which includes at least onecomplement of the coding tag sequence). In this case, the coding tagsare tandemly repeated in the tandem sequence DNA.

Oligonucleotide coding tags can each be any length that supportsspecific and stable hybridization between the coding tags and thedecoding tags. For this purpose, a length of 10 to 35 nucleotides ispreferred, with a coding tag 15 to 20 nucleotides long being mostpreferred.

The branched DNA for use in the disclosed method is generally known(Urdea, Biotechnology 12:926-928 (1994), and Horn et al., Nucleic AcidsRes 23:4835-4841 (1997)). As used herein, the tail of a branched DNAmolecule refers to the portion of a branched DNA molecule that isdesigned to interact with the analyte. The tail is a specific bindingmolecule. In general, each branched DNA molecule should have only onetail. The branches of the branched DNA (also referred to herein as thearms of the branched DNA) contain coding tag sequences. Oligonucleotidedendrimers (or dendrimeric DNA) are also generally known (Shchepinov etal., Nucleic Acids Res. 25:4447-4454 (1997), and Orentas et al., J.Virol. Methods 77:153-163 (1999)). As used herein, the tail of anoligonucleotide dendrimer refers to the portion of a dendrimer that isdesigned to interact with the analyte. In general, each dendrimer shouldhave only one tail. The dendrimeric strands of the dendrimer arereferred to herein as the arms of the oligonucleotide dendrimer andcontain coding tag sequences.

Coding tags can be coupled (directly or via a linker or spacer) toanalytes or other molecules to be labeled. Coding tags can also beassociated with analytes and other molecules to be labeled. For thispurpose, coding molecules are preferred. Coding molecules are moleculesthat can interact with an analyte and with a decoding tag. Codingmolecules include a specific binding molecule and a coding tag. Specificbinding molecules are described above.

N. Reporter Carriers and Coding Carriers

Reporter carriers are associations of one or more specific bindingmolecules, a carrier, and a plurality of reporter signals. Reportercarriers are used in the disclosed method to associate a large number ofreporter signals with an analyte. Coding carriers are associations ofone or more specific binding molecules, a carrier, and a plurality ofcoding tags. Coding carriers are used in the disclosed method toassociate a large number of coding tags with an analyte. The carrier canbe any molecule or structure that facilitates association of manyreporter signals with a specific binding molecule. Examples includeliposomes, microparticles, nanoparticles, virons, phagmids, and branchedpolymer structures. A general class of carriers are structures andmaterials designed for drug delivery. Many such carriers are known.Liposomes are a preferred form of carrier.

Liposomes are artificial structures primarily composed of phospholipidbilayers. Cholesterol and fatty acids may also be included in thebilayer construction. In some forms of the disclosed method, liposomesserve as carriers for arbitrary reporter signals or coding tags. Bycombining liposome reporter carriers, loaded with arbitrary signals ortags, with methods capable of separating a very large multiplicity ofsignals and tags, it becomes possible to perform highly multiplexedassays.

Liposomes, preferably unilamellar vesicles, are made using establishedprocedures that result in the loading of the interior compartment with avery large number (several thousand) of reporter signals or coding tagmolecules, where the chemical nature of these molecules is well suitedfor detection by a preselected detection method. One specific type ofreporter signal or coding tag preferably is used for each specific typeof liposome carrier.

Each specific type of liposome reporter or coding carrier is associatedwith a specific binding molecule. The association may be direct orindirect. An example of a direct association is a liposome containingcovalently coupled antibodies on the surface of the phospholipidbilayer. An alternative, indirect association composition is a liposomecontaining covalently coupled DNA oligonucleotides of arbitrary sequenceon its surface; these oligonucleotides are designed to recognize, bybase complementarity, specific reporter molecules. The reporter moleculemay comprise an antibody-DNA covalent complex, whereby the DNA portionof this complex can hybridize specifically with the complementarysequence on a liposome reporter carrier. In this fashion, the liposomereporter carrier becomes a generic reagent, which may be associatedindirectly with any desired binding molecule.

The use of liposome reporter carriers can be illustrated with thefollowing example.

1. Liposomes (preferably unilamellar vesicles with an average diameterof 150 to 300 nanometers) are prepared using the extrusion method (Hopeet al., Biochimica et Biophysica Acta, 812:55-65 (1985); MacDonald etal., Biochimica et Biophysica Acta, 1061:297-303 (1991)). Other methodsfor liposome preparation may be used as well.

2. A solution of an oligopeptide, at a concentration 400 micromolar, isused during the preparation of the liposomes, such that the inner volumeof the liposomes is loaded with this specific oligopeptide, which willserve to identify a specific analyte of interest. A liposome with aninternal diameter of 200 nanometers will contain, on the average, 960molecules of the oligopeptide. Three separate preparations of liposomesare extruded, each loaded with a different oligopeptide. Theoligopeptides are chosen such that they have the same mass-to-chargeratio but will break into fragments with different mass-to-charge ratiossuch that they will be readily separable by mass spectrometry.

3. The outer surface of the three liposome preparations is conjugatedwith specific antibodies, as follows: a) the first liposome preparationis reacted with an antibody specific for the p53 tumor suppressor; b)the second liposome preparation is reacted with an antibody specific forthe Bcl-2 oncoprotein; c) the third liposome preparation is reacted withan antibody specific or the Her2/neu membrane receptor. Couplingreactions are performed using standard procedures for the covalentcoupling of antibodies to molecules harboring reactive amino groups(Hendrickson et al., Nucleic Acids Research, 23:522-529 (1995);Hermanson, Bioconjugate techniques, Academic Press, pp.528-569(1996);Scheffold et al., Nature Medicine 1:107-110 (2000)). In the case of theliposomes, the reactive amino groups are those present in thephosphatidyl ethanolamine moieties of the liposomes.

4. A glass slide bearing a standard formaldehyde-fixed histologicalsection is contacted with a mixture of all three liposome preparations,suspended in a buffer containing 30 mM Tris-HCl, pH 7.6, 100 mM SodiumChloride, 1 mM EDTA, 0.1% Bovine serum albumin, in order to allowassociation of the liposomes with the corresponding protein antigenspresent in the fixed tissue. After a one hour incubation, the slides arewashed twice, for 5 minutes, with the same buffer (30 mM Tris-HCl, pH7.6, 100 mM Sodium Chloride, 1 mM EDTA, 0.1% Bovine serum albumin). Theslides are dried with a stream of air.

5. The slides are coated with a thin layer of matrix solution consistingof 10 mg/ml alpha-cyano-4-hydroxycinnamic acid, 0.1% trifluoroaceticacid in a 50:50 mixture of acetonitrile in water. The slides are driedwith a stream of air.

6. The slide is placed on the surface of a MALDI plate, and introducedin a mass spectrometer such as that described in Loboda et al., Designand Performance of a MALDI-QqTOFMass Spectrometer, in 47th ASMSConference, Dallas, Tex. (1999), Loboda et al., Rapid Comm. MassSpectrom. 14(12):1047-1057 (2000), Shevchenko et al., Anal. Chem., 72:2132-2142 (2000), and Krutchinsky et al., J. Am. Soc. Mass Spectrom.,11(6):493-504 (2000).

7. Mass spectra are obtained from defined positions on the slidesurface. The relative amount of each of the three peaks of reportersignal polypeptides is used to determine the relative ratios of theantigens detected by the liposome-detector complexes.

The liposome carrier method is not limited to the detection of analyteson histological sections. Cells obtained by sorting may also be used foranalysis in the disclosed method (Scheffold, A., Assenmacher, M.,Reiners-Schramm, L., Lauster, R., and Radbruch, A., 2000, NatureMedicine 1:107-110).

O. Labeled Proteins

Labeled proteins are proteins or peptides to which one or more reportersignals are attached. Preferably, the reporter signal and the protein orpeptide are covalently coupled or tethered to each other. As usedherein, molecules are coupled when they are covalent joined, directly orindirectly. One form of indirect coupling is via a linker molecule. Thereporter signal can be coupled to the protein or peptide by any suitablecoupling reactions. For example, reporter signals can be covalentlycoupled to proteins through a sulfur-sulfur bond between a cysteine onthe protein and a cysteine on the reporter signal. Many otherchemistries and techniques for coupling compounds to proteins are knownand can be used to couple reporter signals to proteins. For example,coupling can be made using thiols, epoxides, nitrites for thiols, NHSesters, isothiocyantes, isothiocyanates for amines, amines, and alcoholsfor carboxylic acids. Proteins and peptides can also be labeled in vivo.

As used herein, “labeled protein” refers to both proteins and peptidesto which one or more reporter signals are attached. The term labeledprotein refers both to proteins and peptides attached to intact (forexample, unfragmented) reporter signals and to proteins and peptidesattached to modified (for example, fragmented) reporter signals. Thelatter form of labeled proteins are referred to as fragmented labeledproteins. Although the protein portion of a labeled protein can befragmented (for example, by protease digestion), the term fragmentedlabeled protein refers to a labeled protein where the reporter signalhas been fragmented. Isobaric labeled proteins are proteins or peptidesof the same type that are labeled with isobaric reporter signals suchthat a set of the proteins has the same mass-to-charge ratio.

P. Labeled Analytes

Labeled analytes are analytes to which one or more reporter signals areattached. Preferably, the reporter signal and the analyte are covalentlycoupled or tethered to each other. As used herein, molecules are coupledwhen they are covalent joined, directly or indirectly. One form ofindirect coupling is via a linker molecule. The reporter signal can becoupled to the analyte by any suitable coupling reactions. Manychemistries and techniques for coupling compounds are known and can beused to couple reporter signals to analytes. For example, coupling canbe made using thiols, epoxides, nitriles for thiols, NHS esters,isothiocyantes, isothiocyanates for amines, amines, and alcohols forcarboxylic acids.

As used herein, “labeled analyte” refers to analytes to which one ormore reporter signals are attached. The term labeled analyte refers bothto analytes attached to intact (for example, unfragmented) reportersignals and to analytes attached to modified (for example, fragmented)reporter signals. The latter form of labeled proteins are referred to asfragmented labeled analytes. Although the analyte portion of a labeledanalyte can be fragmented, the term fragmented labeled analyte refers toa labeled analyte where the reporter signal has been fragmented.Isobaric labeled analytes are analytes of the same type that are labeledwith isobaric reporter signals such that a set of the analytes has thesame mass-to-charge ratio.

Q. Affinity Tags

An affinity tag is any compound that can be used to separate compoundsor complexes having the affinity tag from those that do not. Preferably,an affinity tag is a compound, such as a ligand or hapten, thatassociates or interacts with another compound, such as ligand-bindingmolecule or an antibody. It is also preferred that such interactionbetween the affinity tag and the capturing component be a specificinteraction, such as between a hapten and an antibody or a ligand and aligand-binding molecule. Affinity tags preferably are antibodies,ligands, binding proteins, receptor proteins, haptens, aptamers,carbohydrates, synthetic polyamides, or oligonucleotides. Preferredbinding proteins are DNA binding proteins. Preferred DNA bindingproteins are zinc finger motifs, leucine zipper motifs, helix-turn-helixmotifs. These motifs can be combined in the same specific bindingmolecule.

Affinity tags, described in the context of nucleic acid probes, aredescribed by Syvnen et al., Nucleic Acids Res., 14:5037 (1986).Preferred affinity tags include biotin, which can be incorporated intonucleic acids. In the disclosed method, affinity tags incorporated intoreporter signals can allow the reporter signals to be captured by,adhered to, or coupled to a substrate. Such capture allows separation ofreporter signals from other molecules, simplified washing and handlingof reporter signals, and allows automation of all or part of the method.

Zinc fingers can also be used as affinity tags. Properties of zincfingers, zinc finger motifs, and their interactions, are described byNardelli et al., Zinc finger-DNA recognition: analysis of basespecificity by site-directed mutagenesis. Nucleic Acids Res,20(16):4137-44 (1992), Jamieson et al., In vitro selection of zincfingers with altered DNA-binding specificity. Biochemistry,33(19):5689-95 (1994), Chandrasegaran, S. and J. Smith, Chimericrestriction enzymes: what is next? Biol Chem, 380(7-8):841-8 (1999), andSmith et al., A detailed study of the substrate specificity of achimeric restriction enzyme. Nucleic Acids Res, 27(2):674-81 (1999).

Capturing reporter signals on a substrate, if desired, may beaccomplished in several ways. In one embodiment, affinity docks areadhered or coupled to the substrate. Affinity docks are compounds ormoieties that mediate adherence of a reporter signal by associating orinteracting with an affinity tag on the reporter signal. Affinity docksimmobilized on a substrate allow capture of the reporter signals on thesubstrate. Such capture provides a convenient means of washing awaymolecules that might interfere with subsequent steps. Captured reportersignals can also be released from the substrate. This can beaccomplished by dissociating the affinity tag or by breaking aphotocleavable linkage between the reporter signal and the substrate.

Substrates for use in the disclosed method can include any solidmaterial to which reporter signals can be adhered or coupled. Examplesof substrates include, but are not limited to, materials such asacrylamide, cellulose, nitrocellulose, glass, silicon, polystyrene,polyethylene vinyl acetate, polypropylene, polymethacrylate,polyethylene, polyethylene oxide, polysilicates, polycarbonates, teflon,fluorocarbons, nylon, silicon rubber, polyanhydrides, polyglycolic acid,polylactic acid, polyorthoesters, polypropylfumerate, collagen,glycosaminoglycans, and polyamino acids. Substrates can have any usefulform including thin films or membranes, beads, bottles, dishes, fibers,optical fibers, woven fibers, shaped polymers, particles, compact disks,and microparticles.

R. Vectors and Expression Sequences

Gene transfer can be obtained using direct transfer of genetic material,in but not limited to, plasmids, viral vectors, viral nucleic acids,phage nucleic acids, phages, cosmids, and artificial chromosomes, or viatransfer of genetic material in cells or carriers such as cationicliposomes. Such methods are well known in the art and readily adaptablefor use in the method described herein. Transfer vectors can be anynucleotide construction used to deliver genes into cells (e.g., aplasmid), or as part of a general strategy to deliver genes, e.g., aspart of recombinant retrovirus or adenovirus (Ram et al. Cancer Res.53:83-88, (1993)). Appropriate means for transfection, including viralvectors, chemical transfectants, or physico-mechanical methods such aselectroporation and direct diffusion of DNA, are described by, forexample, Wolff, J. A., et al., Science, 247, 1465-1468, (1990); andWolff, J. A. Nature, 352, 815-818, (1991).

As used herein, plasmid or viral vectors are agents that transport thegene into the cell without degradation and include a promoter yieldingexpression of the gene in the cells into which it is delivered. In apreferred embodiment vectors are derived from either a virus or aretrovirus. Preferred viral vectors are Adenovirus, Adeno-associatedvirus, Herpes virus, Vaccinia virus, Polio virus, AIDS virus, neuronaltrophic virus, Sindbis and other RNA viruses, including these viruseswith the HIV backbone. Also preferred are any viral families which sharethe properties of these viruses which make them suitable for use asvectors. Preferred retroviruses include Murine Maloney Leukemia virus,MMLV, and retroviruses that express the desirable properties of MMLV asa vector. Retroviral vectors are able to carry a larger genetic payload,i.e., a transgene or marker gene, than other viral vectors, and for thisreason are a commonly used vector. However, they are not useful innon-proliferating cells. Adenovirus vectors are relatively stable andeasy to work with, have high titers, and can be delivered in aerosolformulation, and can transfect non-dividing cells. Pox viral vectors arelarge and have several sites for inserting genes, they are thermostableand can be stored at room temperature. A preferred embodiment is a viralvector which has been engineered so as to suppress the immune responseof the host organism, elicited by the viral antigens. Preferred vectorsof this type will carry coding regions for Interleukin 8 or 10.

Viral vectors have higher transaction (ability to introduce genes)abilities than do most chemical or physical methods to introduce genesinto cells. Typically, viral vectors contain, nonstructural early genes,structural late genes, an RNA polymerase III transcript, invertedterminal repeats necessary for replication and encapsidation, andpromoters to control the transcription and replication of the viralgenome. When engineered as vectors, viruses typically have one or moreof the early genes removed and a gene or gene/promotor cassette isinserted into the viral genome in place of the removed viral DNA.Constructs of this type can carry up to about 8 kb of foreign geneticmaterial. The necessary functions of the removed early genes aretypically supplied by cell lines which have been engineered to expressthe gene products of the early genes in trans.

1. Retroviral Vectors

A retrovirus is an animal virus belonging to the virus family ofRetroviridae, including any types, subfamilies, genus, or tropisms.Retroviral vectors, in general, are described by Verma, I. M.,Retroviral vectors for gene transfer. In Microbiology-1985, AmericanSociety for Microbiology, pp. 229-232, Washington, (1985), which isincorporated by reference herein. Examples of methods for usingretroviral vectors for gene therapy are described in U.S. Pat. Nos.4,868,116 and 4,980,286; PCT applications WO 90/02806 and WO 89/07136;and Mulligan, (Science 260:926-932 (1993)); the teachings of which areincorporated herein by reference.

A retrovirus is essentially a package which has packed into it nucleicacid cargo. The nucleic acid cargo carries with it a packaging signal,which ensures that the replicated daughter molecules will be efficientlypackaged within the package coat. In addition to the package signal,there are a number of molecules which are needed in cis, for thereplication, and packaging of the replicated virus. Typically aretroviral genome, contains the gag, pol, and env genes which areinvolved in the making of the protein coat. It is the gag, pol, and envgenes which are typically replaced by the foreign DNA that it is to betransferred to the target cell. Retrovirus vectors typically contain apackaging signal for incorporation into the package coat, a sequencewhich signals the start of the gag transcription unit, elementsnecessary for reverse transcription, including a primer binding site tobind the tRNA primer of reverse transcription, terminal repeat sequencesthat guide the switch of RNA strands during DNA synthesis, a purine richsequence 5′ to the 3′ LTR that serve as the priming site for thesynthesis of the second strand of DNA synthesis, and specific sequencesnear the ends of the LTRs that enable the insertion of the DNA state ofthe retrovirus to insert into the host genome. The removal of the gag,pol, and env genes allows for about 8 kb of foreign sequence to beinserted into the viral genome, become reverse transcribed, and uponreplication be packaged into a new retroviral particle. This amount ofnucleic acid is sufficient for the delivery of a one to many genesdepending on the size of each transcript. It is preferable to includeeither positive or negative selectable markers along with other genes inthe insert.

Since the replication machinery and packaging proteins in mostretroviral vectors have been removed (gag, pol, and env), the vectorsare typically generated by placing them into a packaging cell line. Apackaging cell line is a cell line which has been transfected ortransformed with a retrovirus that contains the replication andpackaging machinery, but lacks any packaging signal. When the vectorcarrying the DNA of choice is transfected into these cell lines, thevector containing the gene of interest is replicated and packaged intonew retroviral particles, by the machinery provided in cis by the helpercell. The genomes for the machinery are not packaged because they lackthe necessary signals.

2. Adenoviral Vectors

The construction of replication-defective adenoviruses has beendescribed (Berkner et al., J. Virology 61:1213-1220 (1987); Massie etal., Mol. Cell. Biol. 6:2872-2883 (1986); Haj-Ahmad et al., J. Virology57:267-274 (1986); Davidson et al., J. Virology 61:1226-1239 (1987);Zhang “Generation and identification of recombinant adenovirus byliposome-mediated transfection and PCR analysis” BioTechniques15:868-872 (1993)). The benefit of the use of these viruses as vectorsis that they are limited in the extent to which they can spread to othercell types, since they can replicate within an initial infected cell,but are unable to form new infectious viral particles. Recombinantadenoviruses have been shown to achieve high efficiency gene transferafter direct, in vivo delivery to airway epithelium, hepatocytes,vascular endothelium, CNS parenchyma and a number of other tissue sites(Morsy, J. Clin. Invest. 92:1580-1586 (1993); Kirshenbaum, J. Clin.Invest. 92:381-387 (1993); Roessler, J. Clin. Invest. 92:1085-1092(1993); Moullier, Nature Genetics 4:154-159 (1993); La Salle, Science259:988-990 (1993); Gomez-Foix, J. Biol. Chem. 267:25129-25134 (1992);Rich, Human Gene Therapy 4:461-476 (1993); Zabner, Nature Genetics6:75-83 (1994); Guzman, Circulation Research 73:1201-1207 (1993); Bout,Human Gene Therapy 5:3-10 (1994); Zabner, Cell 75:207-216 (1993);Caillaud, Eur. J. Neuroscience 5:1287-1291 (1993); and Ragot, J. Gen.Virology 74:501-507 (1993)). Recombinant adenoviruses achieve genetransduction by binding to specific cell surface receptors, after whichthe virus is internalized by receptor-mediated endocytosis, in the samemanner as wild type or replication-defective adenovirus (Chardonnet andDales, Virology 40:462-477 (1970); Brown and Burlingham, J. Virology12:386-396 (1973); Svensson and Persson, J. Virology 55:442-449 (1985);Seth, et al., J. Virol. 51:650-655 (1984); Seth, et al., Mol. Cell.Biol. 4:1528-1533 (1984); Varga et al., J. Virology 65:6061-6070 (1991);Wickham et al., Cell 73:309-319 (1993)).

A preferred viral vector is one based on an adenovirus which has had theE1 gene removed and these virons are generated in a cell line such asthe human 293 cell line. In another preferred embodiment both the E1 andE3 genes are removed from the adenovirus genome.

Another type of viral vector is based on an adeno-associated virus(AAV). This defective parvovirus is a preferred vector because it caninfect many cell types and is nonpathogenic to humans. AAV type vectorscan transport about 4 to 5 kb and wild type AAV is known to stablyinsert into chromosome 19. Vectors which contain this site specificintegration property are preferred. An especially preferred embodimentof this type of vector is the P4.1 C vector produced by Avigen, SanFrancisco, Calif., which can contain the herpes simplex virus thymidinekinase gene, HSV-tk, and/or a marker gene, such as the gene encoding thegreen fluorescent protein, GFP.

The inserted genes in viral and retroviral usually contain promoters,and/or enhancers to help control the expression of the desired geneproduct. A promoter is generally a sequence or sequences of DNA thatfunction when in a relatively fixed location in regard to thetranscription start site. A promoter contains core elements required forbasic interaction of RNA polymerase and transcription factors, and maycontain upstream elements and response elements.

3. Viral Promoters and Enhancers

Preferred promoters controlling transcription from vectors in mammalianhost cells may be obtained from various sources, for example, thegenomes of viruses such as: polyoma, Simian Virus 40 (SV40), adenovirus,retroviruses, hepatitis-B virus and most preferably cytomegalovirus, orfrom heterologous mammalian promoters, e.g. beta actin promoter. Theearly and late promoters of the SV40 virus are conveniently obtained asan SV40 restriction fragment which also contains the SV40 viral originof replication (Fiers et al., Nature, 273: 113 (1978)). The immediateearly promoter of the human cytomegalovirus is conveniently obtained asa HindIII E restriction fragment (Greenway, P. J. et al., Gene 18:355-360 (1982)). Of course, promoters from the host cell or relatedspecies also are useful herein.

Enhancer generally refers to a sequence of DNA that functions at nofixed distance from the transcription start site and can be either 5′(Laimins, L. et al., Proc. Natl. Acad. Sci. 78: 993 (1981)) or 3′(Lusky, M. L., et al., Mol. Cell Bio. 3: 1108 (1983)) to thetranscription unit. Furthermore, enhancers can be within an intron(Banerji, J. L. et al., Cell 33: 729 (1983)) as well as within thecoding sequence itself (Osborne, T. F., et al., Mol. Cell Bio. 4: 1293(1984)). They are usually between 10 and 300 bp in length, and theyfunction in cis. Enhancers function to increase transcription fromnearby promoters. Enhancers also often contain response elements thatmediate the regulation of transcription. Promoters can also containresponse elements that mediate the regulation of transcription.Enhancers often determine the regulation of expression of a gene. Whilemany enhancer sequences are now known from mammalian genes (globin,elastase, albumin, α-fetoprotein and insulin), typically one will use anenhancer from a eukaryotic cell virus. Preferred examples are the SV40enhancer on the late side of the replication origin (bp 100-270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers.

The promotor and/or enhancer may be specifically activated either bylight or specific chemical events which trigger their function. Systemscan be regulated by reagents such as tetracycline and dexamethasone.There are also ways to enhance viral vector gene expression by exposureto irradiation, such as gamma irradiation, or alkylating chemotherapydrugs.

It is preferred that the promoter and/or enhancer region act as aconstitutive promoter and/or enhancer to maximize expression of theregion of the transcription unit to be transcribed. It is furtherpreferred that the promoter and/or enhancer region be active in alleukaryotic cell types. A preferred promoter of this type is the CMVpromoter (650 bases). Other preferred promoters are SV40 promoters,cytomegalovirus (full length promoter), and retroviral vector LTF.

It has been shown that all specific regulatory elements can be clonedand used to construct expression vectors that are selectively expressedin specific cell types such as melanoma cells. The glial fibrillaryacetic protein (GFAP) promoter has been used to selectively expressgenes in cells of glial origin.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human or nucleated cells) may also contain sequencesnecessary for the termination of transcription which may affect mRNAexpression. These regions are transcribed as polyadenylated segments inthe untranslated portion of the mRNA encoding tissue factor protein. The3′ untranslated regions also include transcription termination sites. Itis preferred that the transcription unit also contain a polyadenylationregion. One benefit of this region is that it increases the likelihoodthat the transcribed unit will be processed and transported like mRNA.The identification and use of polyadenylation signals in expressionconstructs is well established. It is preferred that homologouspolyadenylation signals be used in the transgene constructs. In apreferred embodiment of the transcription unit, the polyadenylationregion is derived from the SV40 early polyadenylation signal andconsists of about 400 bases. It is also preferred that the transcribedunits contain other standard sequences alone or in combination with theabove sequences improve expression from, or stability of, the construct.

4. Markers

The viral vectors can include nucleic acid sequence encoding a markerproduct. This marker product is used to determine if the gene has beendelivered to the cell and once delivered is being expressed. Preferredmarker genes are the E. Coli lacZ gene which encodes β-galactosidase andgreen fluorescent protein. In some embodiments the marker may be aselectable marker. Examples of suitable selectable markers for mammaliancells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin,neomycin analog G418, hydromycin, and puromycin. When such selectablemarkers are successfully transferred into a mammalian host cell, thetransformed mammalian host cell can survive if placed under selectivepressure. There are two widely used distinct categories of selectiveregimes. The first category is based on a cell's metabolism and the useof a mutant cell line which lacks the ability to grow independent of asupplemented media. Two examples are: CHO DHFR⁻ cells and mouse LTK⁻cells. These cells lack the ability to grow without the addition of suchnutrients as thymidine or hypoxanthine. Because these cells lack certaingenes necessary for a complete nucleotide synthesis pathway, they cannotsurvive unless the missing nucleotides are provided in a supplementedmedia. An alternative to supplementing the media is to introduce anintact DHFR or TK gene into cells lacking the respective genes, thusaltering their growth requirements. Individual cells which were nottransformed with the DHFR or TK gene will not be capable of survival innon-supplemented media.

The second category is dominant selection which refers to a selectionscheme used in any cell type and does not require the use of a mutantcell line. These schemes typically use a drug to arrest growth of a hostcell. Those cells which have a novel gene would express a proteinconveying drug resistance and would survive the selection. Examples ofsuch dominant selection use the drugs neomycin, (Southern P. and Berg,P., J. Molec. Appl. Genet. 1: 327 (1982)), mycophenolic acid, (Mulligan,R. C. and Berg, P. Science 209: 1422 (1980)) or hygromycin, (Sugden, B.et al., Mol. Cell. Biol. 5: 410-413 (1985)). The three examples employbacterial genes under eukaryotic control to convey resistance to theappropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid)or hygromycin, respectively. Others include the neomycin analog G418 andpuramycin.

Forms and Embodiments of the Disclosed Materials

A. Reporter Molecule Labeling

Disclosed are sets of reporter signals comprising a plurality ofreporter signals, wherein the reporter signals have a common property,wherein the common property allows the reporter signals to bedistinguished and/or separated from molecules lacking the commonproperty, wherein the reporter signals can be altered, wherein thealtered forms of each reporter signal can be distinguished from everyother altered form of reporter signal.

Disclosed are sets of reporter signals, wherein the reporter signalscomprise peptides, wherein the peptides have the same mass-to-chargeratio.

Also disclosed are sets wherein the common property is mass-to-chargeratio, wherein the reporter signals are altered by altering their mass,wherein the altered forms of the reporter signals can be distinguishedvia differences in the mass-to-charge ratio of the altered forms ofreporter signals and sets wherein the mass of the reporter signals isaltered by fragmentation.

In addition, sets wherein the set comprises two or more, three or more,four or more, five or more, six or more, seven or more, eight or more,nine or more, ten or more, twenty or more, thirty or more, forty ormore, fifty or more, sixty or more, seventy or more, eighty or more,ninety or more, or one hundred or more different reporter signals arealso disclosed and sets wherein the set comprises ten or more differentreporter signals.

Disclosed are sets of wherein the reporter signals are peptides,oligonucleotides, carbohydrates, polymers, oligopeptides, or peptidenucleic acids.

Also disclosed are sets wherein the reporter signals are associatedwith, or coupled to, specific binding molecules, wherein each reportersignal is associated with, or coupled to, a different specific bindingmolecule.

Also disclosed are sets wherein the reporter signals are associatedwith, or coupled to, decoding tags, wherein each reporter signal isassociated with, or coupled to, a different decoding tag.

Also disclosed are sets wherein the peptides have the same amino acidcomposition.

Also disclosed are sets wherein the peptides have the same amino acidsequence.

Further disclosed are sets wherein each peptide contains a differentdistribution of heavy isotopes.

Further disclosed are sets wherein each peptide has a different aminoacid sequence.

Also disclosed are sets wherein each peptide has a labile or scissilebond in a different location.

Disclosed are kits comprising (a) a set of reporter molecules, whereineach reporter molecule comprises a reporter signal and a decoding tag,wherein the reporter signals have a common property, wherein the commonproperty allows the reporter signals to be distinguished and/orseparated from molecules lacking the common property, wherein thereporter signals can be altered, wherein the altered forms of eachreporter signal can be distinguished from every other altered form ofreporter signal, wherein each different reporter molecule comprises adifferent decoding tag and a different reporter signal, (b) a set ofcoding molecules, wherein each coding molecule comprises a specificbinding molecule and a coding tag, wherein each specific bindingmolecule can interact specifically with a different analyte, whereineach coding tag can interact specifically with a different decoding tag.

B. Reporter Signal Protein Labeling

Disclosed are sets of labeled proteins wherein each labeled proteincomprises a protein or peptide and a reporter signal attached to theprotein or peptide, wherein the reporter signals have a common property,wherein the common property allows the labeled proteins comprising thesame protein or peptide to be distinguished and/or separated frommolecules lacking the common property, wherein the reporter signals canbe altered, wherein the altered forms of each reporter signal can bedistinguished from every other altered form of reporter signal, whereinalteration of the reporter signals alters the labeled proteins, whereinaltered forms of each labeled protein can be distinguished from everyother altered form of labeled protein.

Disclosed are sets of labeled proteins wherein each labeled proteincomprises a protein or peptide and a reporter signal attached to theprotein or peptide, wherein the labeled proteins have a common property,wherein the common property allows the labeled proteins comprising thesame protein or peptide to be distinguished and/or separated frommolecules lacking the common property, wherein the reporter signals canbe altered, wherein the altered forms of each reporter signal can bedistinguished from every other altered form of reporter signal, whereinalteration of the reporter signals alters the labeled proteins, whereinaltered forms of each labeled protein can be distinguished from everyother altered form of labeled protein.

Disclosed are sets of labeled proteins wherein each labeled proteincomprises a protein or peptide and a reporter signal attached to theprotein or peptide, wherein the reporter signals can be altered, whereinthe altered forms of each reporter signal can be distinguished fromevery other altered form of reporter signal, wherein alteration of thereporter signals alters the labeled proteins, wherein altered forms ofeach labeled protein can be distinguished from every other altered formof labeled protein.

Disclosed are sets of labeled proteins wherein each labeled proteincomprises a protein or peptide and a reporter signal attached to theprotein or peptide, wherein the reporter signals have a common property,wherein the common property allows the labeled proteins comprising thesame protein or peptide to be distinguished and/or separated frommolecules lacking the common property.

Also disclosed are sets wherein the common property is mass-to-chargeratio, wherein the reporter signals are altered by altering their mass,wherein the altered forms of the labeled proteins can be distinguishedvia differences in the mass-to-charge ratio of the altered forms ofreporter signals and sets wherein the mass of the reporter signals isaltered by fragmentation.

Further disclosed are sets wherein alteration of the reporter signalsalso alters their charge.

Also disclosed are sets wherein the common property is mass-to-chargeratio, wherein the reporter signals are altered by altering theircharge, wherein the altered forms of the labeled proteins can bedistinguished via differences in the mass-to-charge ratio of the alteredforms of reporter signals.

Further disclosed are sets wherein the set comprises two or more, threeor more, four or more, five or more, six or more, seven or more, eightor more, nine or more, ten or more, twenty or more, thirty or more,forty or more, fifty or more, sixty or more, seventy or more, eighty ormore, ninety or more, or one hundred or more different reporter signals.

Disclosed are sets wherein the set comprises ten or more differentreporter signals and sets wherein the reporter signals are peptides,oligonucleotides, carbohydrates, polymers, oligopeptides, or peptidenucleic acids.

In addition, disclosed are sets wherein the reporter signals are coupledto the proteins or peptides.

Disclosed are sets wherein the common property allows the labeledproteins to be distinguished and/or separated from molecules lacking thecommon property.

Also disclosed are sets wherein the common property is one or moreaffinity tags associated with the reporter signals and sets wherein oneor more affinity tags are associated with the reporter signals.

Disclosed are sets of labeled proteins wherein each labeled proteincomprises a protein or a peptide and a reporter signal attached to theprotein or peptide, wherein the reporter signals comprise peptides,wherein the reporter signal peptides have the same mass-to-charge ratio.

Also disclosed are sets wherein the reporter signal peptides have thesame amino acid composition and sets wherein the reporter signalpeptides have the same amino acid sequence.

Disclosed are sets wherein each reporter signal peptide contains adifferent distribution of heavy isotopes and sets wherein each reportersignal peptide contains a different distribution of substituent groups.

Disclosed are sets wherein each reporter signal peptide has a differentamino acid sequence and sets wherein each reporter signal peptide has alabile or scissile bond in a different location.

Disclosed are sets wherein one or more affinity tags are associated withthe reporter signals.

Disclosed are kits comprising a set of reporter molecules, wherein eachreporter molecule comprises a reporter signal and a coupling tag,wherein the reporter signals have a common property, wherein the commonproperty allows the reporter signals to be distinguished and/orseparated from molecules lacking the common property, wherein thereporter signals can be altered, wherein the altered forms of eachreporter signal can be distinguished from every other altered form ofreporter signal, wherein each different reporter molecule comprises adifferent coupling tag and a different reporter signal.

Disclosed are labeled proteins wherein the labeled protein comprises aprotein or peptide and a reporter signal attached to the protein orpeptide, wherein the labeled protein has a common property, wherein thecommon property allows the labeled protein to be distinguished and/orseparated from molecules lacking the common property, wherein thereporter signal can be altered, wherein alteration of the reportersignals alters the labeled protein, wherein altered form of the labeledprotein can be distinguished from the unaltered form of labeled protein.

C. Reporter Signal Calibrators

Disclosed are sets of reporter signal calibrators, wherein each reportersignal calibrator shares a common property with a target proteinfragment in a set of target protein fragments, wherein the commonproperty allows the target protein fragments and reporter signalcalibrators having the common property to be distinguished and/orseparated from molecules lacking the common property, wherein the targetprotein fragment and reporter signal calibrator that share a commonproperty correspond to each other, wherein the target protein fragmentscan be altered, wherein the altered forms of the target proteinfragments can be distinguished from the other altered forms of thetarget protein fragments, wherein the reporter signal calibrators can bealtered, wherein the altered form of each reporter signal calibrator canbe distinguished from the altered form of the target protein fragmentwith which the reporter signal calibrator shares a common property.

Also disclosed are sets wherein the set includes a predetermined amountof each reporter signal calibrator and sets wherein the amount of atleast two of the reporter signal calibrators is different.

Disclosed are sets wherein the relative amount each reporter signalcalibrator is based on the relative amount of each corresponding targetprotein fragment expected to be in the protein sample.

Also disclosed are sets wherein the amount of each of the reportersignal calibrators is the same.

Disclosed are sets wherein the target protein fragments and reportersignal calibrators can be altered by fragmentation and sets wherein thetarget protein fragments and reporter signal calibrators can be alteredby cleavage at a photocleavable amino acid.

Disclosed are sets wherein the target protein fragments and reportersignal calibrators can be fragmented in a collision cell and setswherein the target protein fragments can be fragmented at anasparagine-proline bond.

Also disclosed are sets wherein the target protein fragments areproduced by protease digestion of the protein sample and sets whereinthe target protein fragments are produced by digestion of the proteinsample with a serine protease.

Also disclosed are sets wherein the serine protease is trypsin and setswherein the target protein fragments are produced by cleavage at aphotocleavable amino acid.

Disclosed are sets wherein the common property is mass-to-charge ratio,wherein the target protein fragments and reporter signal calibrators canbe altered by altering their mass, their charge, or their mass andcharge, wherein the altered forms of the target protein fragments andreporter signal calibrators can be distinguished via differences in themass-to-charge ratio of the altered forms of the target proteinfragments and reporter signal calibrators.

Also disclosed are sets wherein the set of reporter signal calibratorscomprises two or more, three or more, four or more, five or more, six ormore, seven or more, eight or more, nine or more, ten or more, twenty ormore, thirty or more, forty or more, fifty or more, sixty or more,seventy or more, eighty or more, ninety or more, or one hundred or moredifferent reporter signal calibrators.

Disclosed are sets wherein the set of reporter signal calibratorscomprises ten or more different reporter signal calibrators and setswherein the set of target protein fragments comprises two or more, threeor more, four or more, five or more, six or more, seven or more, eightor more, nine or more, ten or more, twenty or more, thirty or more,forty or more, fifty or more, sixty or more, seventy or more, eighty ormore, ninety or more, or one hundred or more different target proteinfragments.

Disclosed are sets wherein the reporter signal calibrators comprisepeptides, wherein the peptides have the same mass-to-charge ratio as thecorresponding target protein fragments.

Also disclosed are sets wherein the peptides have the same amino acidcomposition as the corresponding target protein fragments.

Also disclosed are sets wherein the peptides have the same amino acidsequence as the corresponding target protein fragments.

In addition, sets are disclosed wherein each peptide has a differentamino acid sequence than the corresponding target protein fragment.

Furthermore, sets are disclosed wherein each peptide has a labile orscissile bond in a different location.

Disclosed are sets wherein the reporter signal calibrators are peptides,oligonucleotides, carbohydrates, polymers, oligopeptides, or peptidenucleic acids and sets wherein at least one of the target proteinfragments comprises at least one modified amino acid.

Also disclosed are sets wherein the modified amino acid is aphosphorylated amino acid, an acylated amino acid, or a glycosylatedamino acid.

Also disclosed are sets wherein at least one of the target proteinfragments is the same as the target protein fragment comprising themodified amino acid except for the modified amino acid.

Disclosed are kits for producing a protein signature, the kit comprising(a) a set of reporter signal calibrators, wherein each reporter signalcalibrator shares a common property with a target protein fragment in aset of target protein fragments, wherein the common property allows thetarget protein fragments and reporter signal calibrators having thecommon property to be distinguished and/or separated from moleculeslacking the common property, wherein the target protein fragment andreporter signal calibrator that share a common property correspond toeach other, wherein the target protein fragments can be altered, whereinthe altered forms of the target protein fragments can be distinguishedfrom the other altered forms of the target protein fragments, whereinthe reporter signal calibrators can be altered, wherein the altered formof each reporter signal calibrator can be distinguished from the alteredform of the target protein fragment with which the reporter signalcalibrator shares a common property, (b) one or more reagents fortreating a protein sample to produce protein fragments.

Also disclosed are kits wherein the set of reporter signal calibratorsincludes a predetermined amount of each reporter signal calibrator.

Also disclosed are kits wherein the amount of at least two of thereporter signal calibrators is different and kits wherein the relativeamount each reporter signal calibrator is based on the relative amountof each corresponding target protein fragment expected to be in theprotein sample.

Disclosed are kits wherein the amount of each of the reporter signalcalibrators is the same and kits wherein the target protein fragmentsand reporter signal calibrators can be altered by fragmentation.

Also disclosed are kits wherein the target protein fragments andreporter signal calibrators can be altered by cleavage at aphotocleavable amino acid and kits wherein the target protein fragmentsand reporter signal calibrators can be fragmented in a collision cell.

Disclosed are kits wherein the target protein fragments can befragmented at an asparagine-proline bond and kits wherein the targetprotein fragments are produced by protease digestion of the proteinsample.

Disclosed are kits wherein the target protein fragments are produced bydigestion of the protein sample with a serine protease and kits whereinthe serine protease is trypsin.

Disclosed are kits wherein the target protein fragments are produced bycleavage at a photocleavable amino acid.

Also disclosed are kits wherein the common property is mass-to-chargeratio, wherein the target protein fragments and reporter signalcalibrators can be altered by altering their mass, their charge, ortheir mass and charge, wherein the altered forms of the target proteinfragments and reporter signal calibrators can be distinguished viadifferences in the mass-to-charge ratio of the altered forms of thetarget protein fragments and reporter signal calibrators.

Disclosed are kits wherein the set of reporter signal calibratorscomprises two or more, three or more, four or more, five or more, six ormore, seven or more, eight or more, nine or more, ten or more, twenty ormore, thirty or more, forty or more, fifty or more, sixty or more,seventy or more, eighty or more, ninety or more, or one hundred or moredifferent reporter signal calibrators.

Also disclosed are kits wherein the set of reporter signal calibratorscomprises ten or more different reporter signal calibrators.

Disclosed are kits wherein the set of target protein fragments comprisestwo or more, three or more, four or more, five or more, six or more,seven or more, eight or more, nine or more, ten or more, twenty or more,thirty or more, forty or more, fifty or more, sixty or more, seventy ormore, eighty or more, ninety or more, or one hundred or more differenttarget protein fragments.

Also disclosed are kits wherein the reporter signal calibrators comprisepeptides, wherein the peptides have the same mass-to-charge ratio as thecorresponding target protein fragments.

Disclosed are kits wherein the peptides have the same amino acidcomposition as the corresponding target protein fragments and kitswherein the peptides have the same amino acid sequence as thecorresponding target protein fragments and kits wherein each peptide hasa different amino acid sequence than the corresponding target proteinfragment.

Disclosed are kits wherein each peptide has a labile or scissile bond ina different location.

Also disclosed are kits wherein the reporter signal calibrators arepeptides, oligonucleotides, carbohydrates, polymers, oligopeptides, orpeptide nucleic acids.

Disclosed are kits wherein at least one of the target protein fragmentscomprises at least one modified amino acid and disclosed are kitswherein the modified amino acid is a phosphorylated amino acid, anacylated amino acid, or a glycosylated amino acid.

Also disclosed are kits wherein at least one of the target proteinfragments is the same as the target protein fragment comprising themodified amino acid except for the modified amino acid.

Disclosed are mixtures comprising a set of reporter signal calibratorsand a set of target protein fragments, wherein each reporter signalcalibrator shares a common property with a target protein fragment inthe set of target protein fragments, wherein the common property allowsthe target protein fragments and reporter signal calibrators having thecommon property to be distinguished and/or separated from moleculeslacking the common property, wherein the target protein fragment andreporter signal calibrator that share a common property correspond toeach other, wherein the target protein fragments can be altered, whereinthe altered forms of the target protein fragments can be distinguishedfrom the other altered forms of the target protein fragments, whereinthe reporter signal calibrators can be altered, wherein the altered formof each reporter signal calibrator can be distinguished from the alteredform of the target protein fragment with which the reporter signalcalibrator shares a common property.

Also disclosed are mixtures wherein the set of reporter signalcalibrators includes a predetermined amount of each reporter signalcalibrator.

Also disclosed are mixtures wherein the amount of at least two of thereporter signal calibrators is different and mixtures wherein therelative amount each reporter signal calibrator is based on the relativeamount of each corresponding target protein fragment expected to be inthe protein sample.

Disclosed are mixtures wherein the amount of each of the reporter signalcalibrators is the same and mixtures wherein the target proteinfragments and reporter signal calibrators can be altered byfragmentation.

Disclosed are mixtures wherein the target protein fragments and reportersignal calibrators can be altered by cleavage at a photocleavable aminoacid.

Also disclosed are mixtures wherein the target protein fragments andreporter signal calibrators can be fragmented in a collision cell.

Disclosed are mixtures wherein the target protein fragments can befragmented at an asparagine-proline bond and mixtures wherein the targetprotein fragments are produced by protease digestion of the proteinsample and mixtures wherein the target protein fragments are produced bydigestion of the protein sample with a serine protease and mixtureswherein the serine protease is trypsin.

Disclosed are mixtures wherein the target protein fragments are producedby cleavage at a photocleavable amino acid and mixtures wherein thecommon property is mass-to-charge ratio, wherein the target proteinfragments and reporter signal calibrators can be altered by alteringtheir mass, their charge, or their mass and charge, wherein the alteredforms of the target protein fragments and reporter signal calibratorscan be distinguished via differences in the mass-to-charge ratio of thealtered forms of the target protein fragments and reporter signalcalibrators.

Disclosed are mixtures wherein the set of reporter signal calibratorscomprises two or more, three or more, four or more, five or more, six ormore, seven or more, eight or more, nine or more, ten or more, twenty ormore, thirty or more, forty or more, fifty or more, sixty or more,seventy or more, eighty or more, ninety or more, or one hundred or moredifferent reporter signal calibrators.

Also disclosed are mixtures wherein the set of reporter signalcalibrators comprises ten or more different reporter signal calibratorsand mixtures wherein the set of target protein fragments comprises twoor more, three or more, four or more, five or more, six or more, sevenor more, eight or more, nine or more, ten or more, twenty or more,thirty or more, forty or more, fifty or more, sixty or more, seventy ormore, eighty or more, ninety or more, or one hundred or more differenttarget protein fragments.

Disclosed are mixtures wherein the reporter signal calibrators comprisepeptides, wherein the peptides have the same mass-to-charge ratio as thecorresponding target protein fragments and mixtures wherein the peptideshave the same amino acid composition as the corresponding target proteinfragments.

Disclosed are mixtures wherein the peptides have the same amino acidsequence as the corresponding target protein fragments and mixtureswherein each peptide has a different amino acid sequence than thecorresponding target protein fragment.

Disclosed are mixtures wherein each peptide has a labile or scissilebond in a different location.

Disclosed are mixtures wherein the reporter signal calibrators arepeptides, oligonucleotides, carbohydrates, polymers, oligopeptides, orpeptide nucleic acids.

Also disclosed are mixtures wherein at least one of the target proteinfragments comprises at least one modified amino acid and mixtureswherein the modified amino acid is a phosphorylated amino acid, anacylated amino acid, or a glycosylated amino acid and mixtures whereinat least one of the target protein fragments is the same as the targetprotein fragment comprising the modified amino acid except for themodified amino acid.

Disclosed are sets of target protein fragments, wherein each targetprotein fragment shares a common property with a reporter signalcalibrator in a set of reporter signal calibrators, wherein the commonproperty allows the target protein fragments and reporter signalcalibrators having the common property to be distinguished and/orseparated from molecules lacking the common property, wherein the targetprotein fragment and reporter signal calibrator that share a commonproperty correspond to each other, wherein the target protein fragmentscan be altered, wherein the altered forms of the target proteinfragments can be distinguished from the other altered forms of thetarget protein fragments, wherein the reporter signal calibrators can bealtered, wherein the altered form of each reporter signal calibrator canbe distinguished from the altered form of the target protein fragmentwith which the reporter signal calibrator shares a common property.

Also disclosed are sets wherein the set of reporter signal calibratorsincludes a predetermined amount of each reporter signal calibrator.

Disclosed are sets wherein the amount of at least two of the reportersignal calibrators is different and sets wherein the relative amounteach reporter signal calibrator is based on the relative amount of eachcorresponding target protein fragment expected to be in the proteinsample.

Disclosed are sets wherein the amount of each of the reporter signalcalibrators is the same and sets wherein the target protein fragmentsand reporter signal calibrators can be altered by fragmentation.

Disclosed are sets wherein the target protein fragments and reportersignal calibrators can be altered by cleavage at a photocleavable aminoacid and sets wherein the target protein fragments and reporter signalcalibrators can be fragmented in a collision cell.

Disclosed are sets wherein the target protein fragments can befragmented at an asparagine-proline bond.

Also disclosed are sets wherein the target protein fragments areproduced by protease digestion of the protein sample.

Also disclosed are sets wherein the target protein fragments areproduced by digestion of the protein sample with a serine protease andsets wherein the serine protease is trypsin and sets wherein the targetprotein fragments are produced by cleavage at a photocleavable aminoacid.

Disclosed are sets wherein the common property is mass-to-charge ratio,wherein the target protein fragments and reporter signal calibrators canbe altered by altering their mass, their charge, or their mass andcharge, wherein the altered forms of the target protein fragments andreporter signal calibrators can be distinguished via differences in themass-to-charge ratio of the altered forms of the target proteinfragments and reporter signal calibrators.

Also disclosed are sets wherein the set of reporter signal calibratorscomprises two or more, three or more, four or more, five or more, six ormore, seven or more, eight or more, nine or more, ten or more, twenty ormore, thirty or more, forty or more, fifty or more, sixty or more,seventy or more, eighty or more, ninety or more, or one hundred or moredifferent reporter signal calibrators.

Also disclosed are sets wherein the set of reporter signal calibratorscomprises ten or more different reporter signal calibrators.

Also disclosed are sets wherein the set of target protein fragmentscomprises two or more, three or more, four or more, five or more, six ormore, seven or more, eight or more, nine or more, ten or more, twenty ormore, thirty or more, forty or more, fifty or more, sixty or more,seventy or more, eighty or more, ninety or more, or one hundred or moredifferent target protein fragments.

Disclosed are sets wherein the reporter signal calibrators comprisepeptides, wherein the peptides have the same mass-to-charge ratio as thecorresponding target protein fragments.

Also disclosed are sets wherein the peptides have the same amino acidcomposition as the corresponding target protein fragments.

Disclosed are sets wherein the peptides have the same amino acidsequence as the corresponding target protein fragments and sets whereineach peptide has a different amino acid sequence than the correspondingtarget protein fragment.

Also disclosed are sets wherein each peptide has a labile or scissilebond in a different location.

Disclosed are sets wherein the reporter signal calibrators are peptides,oligonucleotides, carbohydrates, polymers, oligopeptides, or peptidenucleic acids.

Also disclosed are sets wherein at least one of the target proteinfragments comprises at least one modified amino acid.

Also disclosed are sets wherein the modified amino acid is aphosphorylated amino acid, an acylated amino acid, or a glycosylatedamino acid.

Disclosed are sets wherein at least one of the target protein fragmentsis the same as the target protein fragment comprising the modified aminoacid except for the modified amino acid.

Disclosed are sets of reporter signal calibrators, wherein each reportersignal calibrator shares a common property with a target proteinfragment in a set of target protein fragments, wherein the commonproperty allows each of the target protein fragments and reporter signalcalibrators having the common property to be distinguished and/orseparated from molecules lacking the common property, wherein the targetprotein fragment and reporter signal calibrator that share a commonproperty correspond to each other, wherein each of the target proteinfragments can be altered, wherein the altered forms of each targetprotein fragment can be distinguished from every other altered form oftarget protein fragment, wherein each of the reporter signal calibratorscan be altered, wherein the altered form of each reporter signalcalibrator can be distinguished from the altered form of the targetprotein fragment with which the reporter signal calibrator shares acommon property.

Disclosed are kits for producing a protein signature, the kit comprising(a) a set of reporter signal calibrators, wherein each reporter signalcalibrator shares a common property with a target protein fragment in aset of target protein fragments, wherein the common property allows eachof the target protein fragments and reporter signal calibrators havingthe common property to be distinguished and/or separated from moleculeslacking the common property, wherein the target protein fragment andreporter signal calibrator that share a common property correspond toeach other, wherein each of the target protein fragments can be altered,wherein the altered forms of each target protein fragment can bedistinguished from every other altered form of target protein fragment,wherein each of the reporter signal calibrators can be altered, whereinthe altered form of each reporter signal calibrator can be distinguishedfrom the altered form of the target protein fragment with which thereporter signal calibrator shares a common property, (b) one or morereagents for treating a protein sample to produce protein fragments.

Disclosed are mixtures comprising a set of reporter signal calibratorsand a set of target protein fragments, wherein each reporter signalcalibrator shares a common property with a target protein fragment inthe set of target protein fragments, wherein the common property allowseach of the target protein fragments and reporter signal calibratorshaving the common property to be distinguished and/or separated frommolecules lacking the common property, wherein the target proteinfragment and reporter signal calibrator that share a common propertycorrespond to each other, wherein each of the target protein fragmentscan be altered, wherein the altered forms of each target proteinfragment can be distinguished from every other altered form of targetprotein fragment, wherein each of the reporter signal calibrators can bealtered, wherein the altered form of each reporter signal calibrator canbe distinguished from the altered form of the target protein fragmentwith which the reporter signal calibrator shares a common property.

Also disclosed are sets of target protein fragments, wherein each targetprotein fragment shares a common property with a reporter signalcalibrator in a set of reporter signal calibrators, wherein the commonproperty allows each of the target protein fragments and reporter signalcalibrators having the common property to be distinguished and/orseparated from molecules lacking the common property, wherein the targetprotein fragment and reporter signal calibrator that share a commonproperty correspond to each other, wherein each of the target proteinfragments can be altered, wherein the altered forms of each targetprotein fragment can be distinguished from every other altered form oftarget protein fragment, wherein each of the reporter signal calibratorscan be altered, wherein the altered form of each reporter signalcalibrator can be distinguished from the altered form of the targetprotein fragment with which the reporter signal calibrator shares acommon property.

D. Reporter Signal Fusions

Disclosed are sets of nucleic acid molecules wherein each nucleic acidmolecule comprises a nucleotide segment encoding an amino acid segmentcomprising a reporter signal peptide and a protein or peptide ofinterest, wherein the reporter signal peptides have a common property,wherein the common property allows the reporter signal peptides to bedistinguished and/or separated from molecules lacking the commonproperty, wherein the reporter signal peptides can be altered, whereinthe altered form of each reporter signal peptide can be distinguishedfrom the altered forms of the other reporter signal peptides.

Also disclosed are sets wherein each nucleic acid molecule furthercomprises expression sequences, wherein the expression sequences areoperably linked to the nucleotide segment such that the amino acidsegment can be expressed.

Also disclosed are sets wherein the expression sequences comprisetranslation expression sequences and sets wherein the expressionsequences further comprise transcription expression sequences.

Disclosed are sets wherein the amino acid segment can be expressed invitro and sets wherein the amino acid segment can be expressed in vivoand sets wherein the amino acid segment can be expressed in cellculture.

Also disclosed are sets wherein the expression sequences of each nucleicacid molecule are different and sets wherein the different expressionsequences are differently regulated and sets wherein the expressionsequences are similarly regulated and sets wherein a plurality of theexpression sequences are expression sequences of, or derived from, genesexpressed as part of the same expression cascade and sets wherein theexpression sequences of each nucleic acid molecule are the same and setswherein the expression sequences are similarly regulated and setswherein the expression sequences of at least two nucleic acid moleculesare different and sets wherein the expression sequences of at least twonucleic acid molecules are the same.

Disclosed are sets wherein each nucleic acid molecule further comprisesreplication sequences, wherein the replication sequences allowreplication of the nucleic acid molecules.

Disclosed are sets wherein the nucleic acid molecules can be replicatedin vitro and sets wherein the nucleic acid molecules can be replicatedin vivo and sets wherein the nucleic acid molecules can be replicated incell culture.

Disclosed are sets wherein each nucleic acid molecule further comprisesintegration sequences, wherein the integration sequences allowintegration of the nucleic acid molecules into other nucleic acids.

Also disclosed are sets wherein the nucleic acid molecules can beintegrated into a chromosome and sets wherein the nucleic acid moleculescan be integrated into a chromosome at a predetermined location.

Also disclosed are sets wherein the nucleic acids molecules are producedby replicating nucleic acids in one or more nucleic acid samples.

Also disclosed are sets wherein the nucleic acids are replicated usingpairs of primers, wherein each of the first primers in the primer pairsused to produce the nucleic acid molecules comprises a nucleotidesequence encoding the reporter signal peptide and sets wherein eachfirst primer further comprises expression sequences and sets wherein thenucleotide sequence of each first primer also encodes an epitope tag.

Disclosed are sets wherein each amino acid segment further comprises anepitope tag and sets wherein the epitope tag of each amino acid segmentis different and sets wherein the epitope tag of each amino acid segmentis the same and sets wherein the epitope tag of at least two amino acidsegments are different and sets wherein the epitope tag of at least twoamino acid segments are the same.

Disclosed are sets wherein the reporter signal peptide of each aminoacid segment is different and sets wherein the reporter signal peptideof each amino acid segment is the same and sets wherein the reportersignal peptide of at least two amino acid segments are different andsets wherein the reporter signal peptide of at least two amino acidsegments are the same.

Disclosed are sets wherein the nucleic acid molecules are in cells andsets wherein each nucleic acid molecule is in a different cell and setswherein each nucleic acid molecule is in the same cell and sets whereineach nucleic acid molecule further comprises expression sequences,wherein the expression sequences are operably linked to the nucleotidesegment such that the amino acid segment can be expressed and setswherein the expression sequences of each nucleic acid molecule aredifferent and sets wherein the expression sequences are similarlyregulated and sets wherein a plurality of the expression sequences areexpression sequences of, or derived from, genes expressed as part of thesame expression cascade.

Disclosed are sets wherein the nucleic acid molecules are integratedinto a chromosome of the cell.

Also disclosed are sets wherein the nucleic acid molecules areintegrated into the chromosome at a predetermined location.

Disclosed are sets wherein the chromosome is an artificial chromosome.

Disclosed are sets wherein the nucleic acid molecules are, or areintegrated into, a plasmid.

Also disclosed are sets wherein the cells are in cell lines.

Also disclosed are sets wherein each nucleic acid molecule is in adifferent cell line and sets wherein each nucleic acid molecule is inthe same cell line.

Disclosed are sets wherein the nucleic acid molecules are in organismsand sets wherein each nucleic acid molecule is in a different organismand sets wherein each nucleic acid molecule is in the same organism andsets wherein each nucleic acid molecule further comprises expressionsequences, wherein the expression sequences are operably linked to thenucleotide segment such that the amino acid segment can be expressed andsets wherein the expression sequences of each nucleic acid molecule aredifferent and sets wherein the expression sequences are similarlyregulated and sets wherein a plurality of the expression sequences areexpression sequences of, or derived from, genes expressed as part of thesame expression cascade.

Also disclosed are sets wherein the nucleic acid molecules areintegrated into a chromosome of the organism.

Disclosed are sets wherein the nucleic acid molecules are integratedinto the chromosome at a predetermined location.

Also disclosed are sets wherein the chromosome is an artificialchromosome.

Disclosed are sets wherein the nucleic acid molecules are, or areintegrated into, a plasmid.

Also disclosed are sets wherein each nucleic acid molecule is in adifferent organism and sets wherein each nucleic acid molecule is in thesame organism and sets wherein the nucleic acid molecules are in cellsof an organism and sets wherein the nucleic acid molecules are insubstantially all of the cells of the organism and sets wherein thenucleic acid molecules are in some of the cells of the organism.

Disclosed are sets wherein the amino acid segments are expressed insubstantially all of the cells of the organism and sets wherein theamino acid segments are expressed in some of the cells of the organism.

Disclosed are sets wherein the protein or peptide of interest of eachamino acid segment is different and sets wherein the protein or peptideof interest of each amino acid segment is the same and sets wherein theprotein or peptide of interest of at least two amino acid segments aredifferent and sets wherein the protein or peptide of interest of atleast two amino acid segments are the same and sets wherein the proteinsor peptides of interest are related and sets wherein the proteins orpeptides of interest are proteins produced in the same cascade and setswherein the proteins or peptides of interest are proteins expressedunder the same conditions and sets wherein the proteins or peptides ofinterest are proteins associated with the same disease and sets whereinthe proteins or peptides of interest are proteins associated with thesame cell type and sets wherein the proteins or peptides of interest areproteins associated with the same tissue type and sets wherein theproteins or peptides of interest are proteins in the same enzymaticpathway.

Disclosed are sets wherein the nucleotide segment encodes a plurality ofamino acid segments each comprising a reporter signal peptide and aprotein or peptide of interest and sets wherein the protein or peptideof interest of at least two of the amino acid segments in one of thenucleotide segments are different and sets wherein the protein orpeptide of interest of the amino acid segments in one of the nucleotidesegments are different and sets wherein the protein or peptide ofinterest of at least two of the amino acid segments in each of thenucleotide segments are different and sets wherein the protein orpeptide of interest of the amino acid segments in each of the nucleotidesegments are different.

Disclosed are sets wherein the set consists of a single nucleic acidmolecule and sets wherein the set consists of a single nucleic acidmolecule, wherein the nucleic acid molecule comprises a plurality ofnucleotide segments each encoding an amino acid segment and sets whereinthe amino acid segment comprises a cleavage site near the junctionbetween the reporter signal peptide and the protein or peptide ofinterest and sets wherein the cleavage site is a trypsin cleavage siteand sets wherein the cleavage site is at the junction between thereporter signal peptide and the protein or peptide of interest.

Disclosed are sets wherein each amino acid segment further comprises aself-cleaving segment.

Disclosed are sets wherein the self-cleaving segment is between thereporter signal peptide and the protein or peptide of interest.

Disclosed are sets wherein the self-cleaving segment is an inteinsegment.

Disclosed are sets of nucleic acid molecules wherein each nucleic acidmolecule comprises a nucleotide segment encoding an amino acid segmentcomprising a reporter signal peptide and a protein or peptide ofinterest, wherein the reporter signal peptides have a common property,wherein the common property allows the reporter signal peptides to bedistinguished and/or separated from molecules lacking the commonproperty, wherein the reporter signal peptides can be altered, whereinalteration of the reporter signal peptides alters the amino acidsegments, wherein the altered form of each amino acid segment can bedistinguished from the altered forms of the other amino acid segments.

Disclosed are sets of nucleic acid molecules wherein each nucleic acidmolecule comprises a nucleotide segment encoding an amino acid segmentcomprising a reporter signal peptide and a protein or peptide ofinterest, wherein the amino acid segments have a common property,wherein the common property allows the amino acid segments to bedistinguished and/or separated from molecules lacking the commonproperty, wherein the reporter signal peptides can be altered, whereinthe altered form of each reporter signal peptide can be distinguishedfrom the altered forms of the other reporter signal peptides.

Disclosed are sets of nucleic acid molecules wherein each nucleic acidmolecule comprises a nucleotide segment encoding an amino acid segmentcomprising a reporter signal peptide and a protein or peptide ofinterest, wherein the amino acid segments have a common property,wherein the common property allows the amino acid segments to bedistinguished and/or separated from molecules lacking the commonproperty, wherein the reporter signal peptides can be altered, whereinalteration of the reporter signal peptides alters the amino acidsegments, wherein the altered form of each amino acid segment can bedistinguished from the altered forms of the other amino acid segments.

Disclosed are sets of nucleic acid molecules wherein each nucleic acidmolecule comprises a nucleotide segment encoding an amino acid segmentcomprising a reporter signal peptide and a protein or peptide ofinterest, wherein the amino acid segments each comprise an amino acidsubsegment, wherein each amino acid subsegment comprises a portion ofthe protein or peptide of interest and all or a portion of the reportersignal peptide, wherein the amino acid subsegments have a commonproperty, wherein the common property allows the amino acid subsegmentsto be distinguished and/or separated from molecules lacking the commonproperty, wherein the reporter signal peptides can be altered, whereinthe altered form of each reporter signal peptide can be distinguishedfrom the altered forms of the other reporter signal peptides.

Disclosed are sets of nucleic acid molecules wherein each nucleic acidmolecule comprises a nucleotide segment encoding an amino acid segmentcomprising a reporter signal peptide and a protein or peptide ofinterest, wherein the amino acid segments each comprise an amino acidsubsegment, wherein each amino acid subsegment comprises a portion ofthe protein or peptide of interest and all or a portion of the reportersignal peptide, wherein the amino acid subsegments have a commonproperty, wherein the common property allows the amino acid subsegmentsto be distinguished and/or separated from molecules lacking the commonproperty, wherein the reporter signal peptides can be altered, whereinalteration of the reporter signal peptides alters the amino acidsubsegments, wherein the altered form of each amino acid subsegment canbe distinguished from the altered forms of the other amino acidsubsegments.

Disclosed are sets of amino acid segments wherein each amino acidsegment comprises a reporter signal peptide and a protein or peptide ofinterest, wherein the reporter signal peptides have a common property,wherein the common property allows the reporter signal peptides to bedistinguished and/or separated from molecules lacking the commonproperty, wherein the reporter signal peptides can be altered, whereinthe altered form of each reporter signal peptide can be distinguishedfrom the altered forms of the other reporter signal peptides.

Also disclosed are sets wherein the amino acid segment is a protein orpeptide and sets wherein the set consists of a single amino acidsegment, wherein the amino acid segment comprises a plurality ofreporter signal peptides.

Also disclosed cells comprising a set of nucleic acid molecules whereineach nucleic acid molecule comprises a nucleotide segment encoding anamino acid segment comprising a reporter signal peptide and a protein orpeptide of interest, wherein the reporter signal peptides have a commonproperty, wherein the common property allows the reporter signalpeptides to be distinguished and/or separated from molecules lacking thecommon property, wherein the reporter signal peptides can be altered,wherein the altered form of each reporter signal peptide can bedistinguished from the altered forms of the other reporter signalpeptides.

Disclosed are sets of cells wherein each cell comprises a nucleic acidmolecule wherein each nucleic acid molecule comprises a nucleotidesegment encoding an amino acid segment comprising a reporter signalpeptide and a protein or peptide of interest, wherein the reportersignal peptides have a common property, wherein the common propertyallows the reporter signal peptides to be distinguished and/or separatedfrom molecules lacking the common property, wherein the reporter signalpeptides can be altered, wherein the altered form of each reportersignal peptide can be distinguished from the altered forms of the otherreporter signal peptides.

Also disclosed are sets wherein each cell further comprises additionalnucleic acid molecules and sets wherein the set consists of a singlecell, wherein the cell comprises a plurality of nucleic acid moleculesand sets wherein the set consists of a single cell, wherein the cellcomprises a set of nucleic acid molecules, wherein the set of nucleicacid molecules consists of a single nucleic acid molecule, wherein thenucleic acid molecule encodes a plurality of nucleic acid segments.

Disclosed are organisms comprising a set of nucleic acid moleculeswherein each nucleic acid molecule comprises a nucleotide segmentencoding an amino acid segment comprising a reporter signal peptide anda protein or peptide of interest, wherein the reporter signal peptideshave a common property, wherein the common property allows the reportersignal peptides to be distinguished and/or separated from moleculeslacking the common property, wherein the reporter signal peptides can bealtered, wherein the altered form of each reporter signal peptide can bedistinguished from the altered forms of the other reporter signalpeptides.

Disclosed are sets of organisms each organism comprises a nucleic acidmolecule wherein each nucleic acid molecule comprises a nucleotidesegment encoding an amino acid segment comprising a reporter signalpeptide and a protein or peptide of interest, wherein the reportersignal peptides have a common property, wherein the common propertyallows the reporter signal peptides to be distinguished and/or separatedfrom molecules lacking the common property, wherein the reporter signalpeptides can be altered, wherein the altered form of each reportersignal peptide can be distinguished from the altered forms of the otherreporter signal peptides.

Also disclosed are sets wherein each organism further comprisesadditional nucleic acid molecules and sets wherein the set consists of asingle organism, wherein the organism comprises a plurality of nucleicacid molecules and sets wherein the set consists of a single organism,wherein the organism comprises a set of nucleic acid molecules, whereinthe set of nucleic acid molecules consists of a single nucleic acidmolecule, wherein the nucleic acid molecule encodes a plurality ofnucleic acid segments.

Method

The disclosed methods are useful for sensitive detection of one ormultiple analytes. In general, the methods involve the use of speciallabel components, referred to as reporter signals, that can beassociated with, incorporated into, or otherwise linked to the analytes,or that can be used merely in conjunction with analytes, with nosignificant association between the analytes and reporter signals. Insome embodiments of the methods, the reporter signals (or derivatives ofthe reporter signals) are detected, thus indicating the presence of theassociated analytes. In other embodiments, the analyte (or derivativesof the analytes) are detected along with the reporter signals (orderivatives of the reporter signals).

In some embodiments of the methods, the reporter signals can be alteredsuch that the altered forms of different reporter signals can bedistinguished from each other. Reporter signal/analyte conjugates can bealtered, generally through alteration of the reporter signal portion ofthe conjugate, such that the altered forms of different reportersignals, altered forms of different reporter signal/analyte conjugates,or both, can be distinguished from each other. Where the reporter signalor reporter signal/analyte conjugate is altered by fragmentation, any,some, or all of the fragments can be distinguished from each other,depending on the embodiment. For example, where reporter signalsfragmented into two parts, either or both parts of the reporter signalscan be distinguished. Where reporter signal/analyte conjugates arefragmented into two parts (with the break point in the reporter signalportion), either the reporter signal fragment, the reportersignal/analyte fragment, or both can be distinguished. In someembodiments, only one part of a fragmented reporter signal will bedetected and so only this part of the reported signals need bedistinguished.

In some embodiments of the methods, sets of reporter signals can be usedwhere two or more of the reporter signals in a set have one or morecommon properties that allow the reporter signals having the commonproperty to be distinguished and/or separated from other moleculeslacking the common property. In other embodiments, sets of reportersignal/analyte conjugates can be used where two or more of the reportersignal/analyte conjugates in a set have one or more common propertiesthat allow the reporter signal/analyte conjugates having the commonproperty to be distinguished and/or separated form other moleculeslacking the common property. In still other embodiments, analytes can befragmented (prior to or following conjugation) to produce reportersignal/analyte fragment conjugates (which can be referred to as fragmentconjugates). In such cases, sets of fragment conjugates can be usedwhere two or more of the fragment conjugates in a set have one or morecommon properties that allow the fragment conjugates having the commonproperty to be distinguished and/or separated from other moleculeslacking the common property.

As indicated above, reporter signals conjugated with analytes can bealtered while in the conjugate and distinguished. Conjugated reportersignals can also be dissociated or separated, in whole or in part, fromthe conjugated analytes prior to their alteration. Where the reportersignals are dissociated (in whole or in part) from the analytes, themethod can be performed such that the fact of association between theanalyte and reporter signal is part of the information obtained when thereporter signal is detected. In other words, the fact that the reportersignal may be dissociated from the analyte for detection does notobscure the information that the detected reporter signal was associatedwith the analyte.

Reporter signals can also be in conjunction with analytes (such as inmixtures of reporter signals and analytes), where no significantphysical association between the reporter signals and analytes occurs;or alone, where no analyte is present. In such cases, where reportersignals are not or are no longer associated with analytes, sets ofreporter signals can be used where two or more of the reporter signalsin a set have one or more common properties that allow the reportersignals having the common property to be distinguished and/or separatedfrom other molecules lacking the common property.

In preferred embodiments, the disclosed methods involves two basicsteps. A filtering, selection, or separation step to separate reportersignals from other molecules that may be present, and a detection stepthat distinguishes different reporter signals. The reporter signalspreferably are distinguished and/or separated from other molecules basedon some common property shared by the reporter signals but not presentin most (or, preferably, all) other molecules present. The separatedreporter signals are then treated and/or detected such that thedifferent reporter signals are distinguishable. Useful forms of thedisclosed method involve association of reporter signals with analytesof interest. Detection of the reporter signals results in detection ofthe corresponding analytes. Thus, the disclosed method is a generaltechnique for labeling and detection of analytes.

A preferred form of the disclosed method involves filtering of isobaricreporter signals from other molecules based on mass-to-charge ratio,fragmentation of the reporter signals to produce fragments havingdifferent masses, and detection of the different fragments based ontheir mass-to-charge ratios. The method is best carried out using atandem mass spectrometer. There are two types of tandem massspectrometers, as well as hybrids and combinations of these types:“tandem in space” spectrometers and “tandem in time” spectrometers.Tandem in space spectrometers utilize spatially ordered elements and actupon the ions in turn as the ions pass through each element. Tandem intime spectrometers utilize temporally ordered manipulations on the ionsas the ions are contained in a space. In a tandem in space class ofinstrument, the isobaric reporter signals are first passed through afiltering quadrupole, the reporter signals are fragmented (preferably ina collision cell), and the fragments are distinguished and detected in atime-of-flight (TOF) stage. In such an instrument the sample is ionizedin the source (for example, in a MALDI) to produce charged ions. It ispreferred that the ionization conditions are such that primarily asingly charged parent ion is produced. A first quadrupole, Q0, isoperated in radio frequency (RF) mode only and acts as an ion guide forall charged particles. The second quadrupole, Q1, is operated in RF+DCmode to pass only a narrow range of mass-to-charge ratios (that includesthe mass-to-charge ratio of the reporter signals). This quadrupoleselects the mass-to-charge ratio of interest. Quadrupole Q2, surroundedby a collision cell, is operated in RF only mode and acts as ion guide.The collision cell surrounding Q2 can be filled to appropriate pressurewith a gas to fracture the input ions by collisionally induceddissociation. The collision gas preferably is chemically inert, butreactive gases can also be used. Preferred molecular systems utilizereporter signals that contain scissile bonds, labile bonds, orcombinations, such that these bonds will be preferentially fractured inthe Q2 collision cell.

The same sample can be analyzed both with and without fragmentation (byoperating with and without collision gas), and the results compared todetect shifts in mass-to-charge ratio. Both the unfragmented andfragmented results should give diagnostic peaks, with the combination ofpeaks both with and without fragmentation confirming the reporter signal(and analyte) involved. Such distinctions are accomplished by usingappropriate sets of isobaric reporter signals and allows large scalemultiplexing in the detection of analytes.

The disclosed method is particularly well suited to the use of aMALDI-QqTOF mass spectrometer. The method enables highly multiplexedanalyte detection, and very high sensitivity. Preferred tandem massspectrometers are described by Loboda et al., Design and Performance ofa MALDI-QqTOF Mass Spectrometer, in 47th ASMS Conference, Dallas, Tex.(1999), Loboda et al., Rapid Comm. Mass Spectrom. 14(12):1047-1057(2000), Shevchenko et al., Anal. Chem., 72: 2132-2142 (2000), andKrutchinsky et al., J. Am. Soc. Mass Spectrom., 11(6):493-504 (2000). Insuch an instrument the sample is ionized in the source (MALDI, forexample) to produce charged ions; it is preferred that the ionizationconditions are such that primarily a singly charged parent ion isproduced. First and third quadrupoles, Q0 and Q2, will be operated in RFonly mode and will act as ion guides for all charged particles, secondquadrupole Q1 will be operated in RF+DC mode to pass only a particularmass-to-charge (or, in practice, a narrow mass-to-charge range). Thisquadrupole selects the mass-to-charge ratio, (m/z), of interest. Thecollision cell surrounding Q2 can be filled to appropriate pressure witha gas to fracture the input ions by collisionally induced dissociation(normally the collision gas is chemically inert, but reactive gases arecontemplated). Preferred molecular systems utilize reporter signals thatcontain scissile bonds, labile bonds, or combinations, and these bondswill be preferentially fractured in the Q2 collision cell.

A MALDI source is preferred for the disclosed method because itfacilitates the multiplexed analysis of samples from heterogeneousenvironments such as arrays, beads, microfabricated devices, tissuesamples, and the like. An example of such an instrument is described byQin et al., A practical ion trap mass spectrometer for the analysis ofpeptides by matrix-assisted laser desorption/ionization., Anal. Chem.,68:1784-1791 (1996). For homogeneous assays electrospray ionization(ESI) sources will work very well. Electrospray ionization sourceinstruments interfaced to LC systems are commercially available (forexample, QSTAR from PE-SCIEX, Q-TOF from Micromass). It is of note thatthe ESI sources are operated such that they tend to produce multiplycharged ions, doubly charged ions would be most common for ions in thedisclosed method. Such doubly charged ions are well known in the art andpresent no limitation to the disclosed method. TOF analyzers andquadrupole analyzers are preferred detectors over sector analyzers.Tandem in time ion trap systems such as Fourier Transform Ion CyclotronResonance (FT-ICR) mass spectrometers also may be used with thedisclosed method.

A number of elements contribute to the sensitivity of the disclosedmethod. The filter quadrupole, Q1, selects a narrow mass-to-charge ratioand discriminates against other mass-to-charge ions, significantlydecreasing background from non germane ions. For example, for a samplecontaining a distribution of mass-to-charges of width 3000 Da, amass-to-charge transmission window of 2 Da applied to this distributioncan improve the signal to noise by at least a factor of 3000/2=1500.Once the parent ion is selected by quadrupole Q1, fragmentation of theparent ion, preferably into a single charged daughter ion, has theadvantage over systems which fragment the parent into a number ofdaughter ions. For example, a parent fragmented into 20 daughter ionswill yield signals that are on average {fraction (1/20)}^(th) theintensity of the parent ions. For a parent to single daughter systemthere will not be this signal dilution.

This preferred system for use with the disclosed method has a high dutycycle, and as such good statistics can be collected quickly. For thecase where a single set of isobaric parents is used, the multiplexeddetection is accomplished without having to scan the filter quadrupole(although such a scan is useful for single pass analysis of a complexprotein sample with multiple labeled proteins). Electrospray sources canoperate continuously, MALDI sources can operate at several kHz,quadrupoles operate continuously, and time of flight analyzers cancapture the entire mass-to-charge region of interest at several kHzrepetition rate. Thus, the overall system can acquire thousands ofmeasurements per second. For throughput advantage in a multiplexed assaythe time of flight analyzer has an advantage over a quadruple analyzerfor the final stage because the time of flight analyzer detects allfragment ions in the same acquisition rather than requiring scanning (orstepping) over the ions with a quadrupole analyzer.

Instrumental improvements including addition of laser ports along theflight path to allow intersection of the proteins with additionallaser(s) open additional fragmentation avenues through photochemical andphotophysical processes (for example, selective bond cleavage, selectiveionization). Use of lasers to fragment the proteins after the filterstage will enable the use of the very high throughput TOF-TOFinstruments (50 kHz to 100 kHz systems).

The disclosed method is compatible with techniques involving cleavage,treatment, or fragmentation of a bulk sample in order to simplify thesample prior to introduction into the first stage of a multistagedetection system. The disclosed method is also compatible with anydesired sample, including raw extracts and fractionated samples.

A. Reporter Molecule Labeling

In one form of the disclosed method, referred to as reporter moleculelabeling, reporter signals are associated with analytes to be detectedand/or quantitated. For example, a reporter signal can be associatedwith a specific binding molecule that interacts with the analyte ofinterest. Such a combination is referred to as a reporter molecule. Thespecific binding molecule in the reporter molecule interacts with theanalyte thus associating the reporter signal with the analyte.Alternatively, a reporter signal can be associated with an analyteindirectly. In this mode, a “coding” molecule containing a specificbinding molecule and a coding tag is associated with the analyte (viathe specific binding molecule). Alternatively, a coding tag can becoupled or directly associated with the analyte. Then a reporter signalassociated with a decoding tag (such a combination is another form ofreporter molecule) is associated with the coding molecule through aninteraction between the coding tag and the decoding tag. An example ofthis interaction is hybridization where the coding and decoding tags arecomplementary nucleic acid sequences. The result is an indirectassociation of the reporter signal with the analyte. This mode has theadvantage that all of the interactions of the reporter signals with thecoding molecule can be made chemically and physically similar by usingthe same types of coding tags and decoding tags for all of the codingmolecules and reporter molecules in a set.

The disclosed method increases the sensitivity and accuracy of detectionof an analyte of interest. Preferred forms of the disclosed method makeuse of multistage detection systems to increase the resolution of thedetection of molecules having very similar properties. The methodinvolves at least two stages. The first stage is filtration or selectionthat allows passage or selection of reporter signals (that is, a subsetof the molecules present), based upon intrinsic properties of thereporter signals, and discrimination against all other molecules. Thesubsequent stage(s) further separate(s) and/or detect(s) the reportersignals which were filtered in the first stage. A key facet of thismethod is that a multiplexed set of reporter signals will be selected bythe filter and subsequently cleaved, decomposed, reacted, or otherwisemodified to realize the identities and/or quantities of the reportersignals in further stages. There is a correspondence between thespecific binding molecule and the detected daughter fragment.

B. Reporter Signal Labeling

Another form of the disclosed method, referred to as reporter signallabeling, involves detection of analytes by detecting a reporter signal,labeled analyte, or both; or by distinguishing different reportersignals, different labeled analytes, or both. Detection of the reportersignals results in detection of the corresponding labeled analytes(where the analytes are labeled with the reporter signals). Detection ofthe labeled analytes results in detection of the corresponding analytes.Thus, reporter signal labeling is a general technique for labeling,detection, and quantitation of analytes.

In one embodiment, the disclosed method can involve two basic steps. Afiltering, selection, or separation step to separate labeled analytesfrom other molecules that may be present, and a detection step thatdetects a reporter signal, labeled analyte, or both; or thatdistinguishes different reporter signals, different labeled analytes, orboth. The labeled analytes preferably are distinguished and/or separatedfrom other molecules based on some common property shared by theattached reporter signals but not present in most (or, preferably, all)other molecules present. The labeled analytes can also be distinguishedand/or separated from other molecules based on a common property of thelabeled analyte as a whole, such as the mass-to-charge ratio of thelabeled analyte. The separated labeled analytes are then treated and/ordetected in such a way that the different reporter signals, differentlabeled analytes, or both, are distinguishable.

Reporter signals can be coupled or directly associated with an analyte.For example, a reporter signal can be coupled to an analyte via reactivegroups, or a reporter molecule (composed of a specific binding moleculeand a reporter signal) can be associated with an analyte. Alternatively,a reporter signal can be associated with an analyte indirectly. In thismode, a “coding” molecule containing a specific binding molecule and acoding tag can be associated with the analyte (via the specific bindingmolecule). Alternatively, a coding tag can be coupled or directlyassociated with the analyte. Then a reporter signal associated with adecoding tag (such a combination is another form of reporter molecule)is associated with the coding molecule through an interaction betweenthe coding tag and the decoding tag. An example of this interaction ishybridization where the coding and decoding tags are complementarynucleic acid sequences. The result is an indirect association of thereporter signal with the analyte. This mode has the advantage that allof the interactions of the reporter signals with the coding molecule canbe made chemically and physically similar by using the same types ofcoding tags and decoding tags for all of the coding molecules andreporter molecules in a set.

Reporter signals, or constructs containing reporters signals, also canbe attached or coupled to analytes by ligation. Methods for ligation ofnucleic acids are well known (see, for example, Sambrook et al.Molecular Cloning: A Laboratory Manual, second edition, 1989, ColdSpring Harbor Laboratory Press, New York.), and efficient proteinligation is known (see, for example, Dawson et al., “Synthesis ofproteins by native chemical ligation” Science 266, 776-9 (1994); Hackenget al., “Chemical synthesis and spontaneous folding of a multidomainprotein: anticoagulant microprotein S” Proc Natl Acad Sci USA 97:14074-8(2000); Dawson et al., “Synthesis of Native Proteins by ChemicalLigation” Ann. Rev. Biochem. 69:923-960 (2000); U.S. Pat. No. 6,184,344;PCT Publication WO 98/28434).

The disclosed method can be used in many modes. For example, thedisclosed method can be used to detect a specific analyte (in a specificsample or in multiple samples) or multiple analytes (in a single sampleor multiple samples). In each case, the analyte(s) to be detected can beseparated either from other, unlabeled analytes or from other moleculeslacking a property of the labeled analyte(s) to be detected. Forexample, analytes in a sample can be generally labeled with reportersignals and some analytes can be separated on the basis of some propertyof the analytes. For example, the separated analytes could have acertain mass-to-charge ratio (separation based on mass-to-charge ratiowill select both labeled and unlabeled analytes having the selectedmass-to-charge ratio). As another example, all of the labeled analytescan be distinguished and/or separated from unlabeled molecules based ona feature of the reporter signal such as an affinity tag. Wheredifferent affinity tags are used, some labeled analytes can bedistinguished and/or separated from others.

In one mode of the disclosed method, multiple analytes in multiplesamples are labeled where all of the analytes in a given sample arelabeled with the same reporter signal. That is, the reporter signal isused as a general label of the analytes in a sample. Each sample,however, uses a different reporter signal. This allows samples as awhole to be compared with each other. By additionally separating ordistinguishing different analytes in the samples, one can easily analyzemany analytes in many samples in a single assay. For example, proteinsin multiple samples can be labeled with reporter signals as describedabove, and the samples mixed together. If some or all of the variouslabeled proteins are separated by, for example, association of theproteins with antibodies on an array, the presence and amount of a givenprotein in each of the samples can be determined by identifying thereporter signals present at each array element. If the proteincorresponding to a given array element was present in a particularsample, then some of the protein associated with that array element willbe labeled with the reporter signal used to label that particularsample. Detection of that reporter signal will indicate this. This samerelationship holds true for all of the other samples. Further, theamount of reporter signal detected can indicate the amount of a givenprotein in a given sample, and the simultaneous quantitation of proteinin multiple samples can provide a particularly accurate comparison ofthe levels of the proteins in the various samples.

Optionally, the selection step can be preceded by fractionation stepwhere a subset of analytes, including the analytes that are, or will be,labeled, are separated from other components in a sample. Such a step,although not necessary, can improve the selection step by reducing thenumber of extraneous molecules present.

A preferred form of the disclosed method involves filtering of isobariclabeled analytes from other molecules based on mass-to-charge ratio,fragmentation of the reporter signals to produce fragments havingdifferent mass-to-charge ratios, and detection of the differentfragments based on their mass-to-charge ratios. The different fragmentswill include the fragment of the reporter signal and the fragmentedlabeled analyte (made up of the analyte and the remaining part of thereporter signal). Either or both may be detected and will becharacteristic of the initial labeled analyte. The method is bestcarried out using a tandem mass spectrometer. In such an instrument theisobaric reporter signals are first filtered, then reporter signals arefragmented (preferably by collision), and the fragments aredistinguished and detected.

A preferred form of the disclosed method involves detection of labeledanalytes in two or more samples in the same assay. This allows simpleand consistent detection of differences between the analytes in thesamples. Differential detection is accomplished by labeling the analytesin each sample with a different reporter signal. Preferably, thedifferent reporter signals used for the different samples will make upan isobaric set. In this way, the same labeled analyte in each samplewill have the same mass-to-charge ratio as that labeled analyte in adifferent sample. Upon fragmentation of the reporter signals, however,each of the fragmented labeled analytes in the different samples willhave a different mass-to-charge ratio and thus each can be separatelydetected. All can be detected in the same measurement. This is atremendous advantage in both time and quality of the data. For example,since the samples are assayed in a single run, there is no need tocorrect or normalize the results of different samples assayed indifferent runs. This allows accurate comparisons of the relative amountsof the same analyte in different samples since that are measured in thesame run. There would be no differences to cause inconsistency betweenthe samples.

A preferred use for this multiple sample mode of the disclosed method isthe analysis of a time series of samples. Such series are useful fordetecting changes in a sample or reaction over time. For example,changes in analyte levels in a cell culture over time after addition ofa test compound can be assessed. In this mode, different time pointsamples are labeled with different reporter signals, preferably makingup an isobaric set. In this way, the same labeled analyte for each timepoint will have the same mass-to-charge ratio as that labeled analytefrom a different time point. Upon fragmentation of the reporter signals,however, each of the fragmented labeled analytes from the different timepoints will have a different mass-to-charge ratio and thus each can beseparately detected.

The disclosed method can also be used to gather and catalog informationabout unknown analytes. This analyte discovery mode can easily link thepresence or pattern of analytes with their analysis. For example, asample of labeled analytes can be compared to analytes in one or moreother samples. Analytes that appear in one or some samples but notothers can be analyzed using conventional techniques. The objectanalytes will be distinguishable from others by virtue of the disclosedlabeling, detection, and quantitation. This mode of the method ispreferably carried out using mass spectrometry.

The disclosed method increases the sensitivity and accuracy of detectionof analytes of interest. Preferred forms of the disclosed method makeuse of multistage detection systems to increase the resolution of thedetection of molecules having very similar properties. The method caninvolve at least two stages. The first stage is filtration or selectionthat allows passage or selection of labeled analytes (that is, a subsetof the molecules present), based upon intrinsic properties of thereporter signals (and the attached analytes), and discrimination againstall other molecules. The subsequent stage(s) further separate(s) and/ordetect(s) the labeled analytes that were filtered in the first stage. Akey facet of this method is that a multiplexed set of labeled analyteswill be selected by the filter and the attached reporter signalssubsequently will be cleaved, decomposed, reacted, or otherwise modifiedto realize the identities and/or quantities of the fragmented reportersignals and/or the fragmented labeled analytes in further stages. Thereis a correspondence between the reporter signal and the detecteddaughter fragment(s).

In some embodiments, the disclosed method allows a complex sample ofanalytes to be quickly and easily cataloged in a reproducible manner.Such a catalog can be compared with other, similarly prepared catalogsof other analyte samples to allow convenient detection of differencesbetween the samples. The catalogs, which incorporate a significantamount of information about the analyte samples, can serve asfingerprints of the samples which can be used both for detection ofrelated analyte samples and comparison of analyte samples. For example,the presence or identity of specific organisms can be detected byproducing a catalog of analytes of the test organism and comparing theresulting catalog with reference catalogs prepared from known organisms.Changes and differences in analyte patterns can also be detected bypreparing catalogs of analytes from different cell samples and comparingthe catalogs. Comparison of analyte catalogs produced with the disclosedmethod is facilitated by the fine resolution that can be provided with,for example, mass spectrometry.

Each labeled analyte processed in the disclosed method will result in asignal based on the characteristics of the labeled analyte (for example,the mass-to-charge ratio). A complex analyte sample can produce a uniquepattern of signals. It is this pattern that can allow unique catalogingof analyte samples and sensitive and powerful comparisons of thepatterns of signals produced from different analyte samples.

The presence, amount, presence and amount, or absence of differentlabeled analytes forms a pattern of signals that provides a signature orfingerprint of the analytes, and thus of the analyte sample based on thepresence or absence of specific analytes or analyte fragments in thesample. For this reason, cataloging of this pattern of signals (that is,the pattern of the presence, amount, presence and amount, or absence oflabeled analytes) is an embodiment of the disclosed method that is ofparticular interest.

Catalogs can be made up of, or be referred to, as, for example, apattern of labeled analytes, a pattern of the presence of labeledanalytes, a catalog of labeled analytes, or a catalog of analytes in asample. The information in the catalog is preferably in the form ofmass-to-charge information or compositional information. Catalogs canalso contain or be made up of other information derived from theinformation generated in the disclosed method (for example, the identityof the analytes detected), and can be combined with information obtainedor generated from any other source. The informational nature of catalogsproduced using the disclosed method lends itself to combination and/oranalysis using known bioinformatics systems and methods.

Such catalogs of analyte samples can be compared to a similar catalogderived from any other sample to detect similarities and differences inthe samples (which is indicative of similarities and differences in theanalytes in the samples). For example, a catalog of a first analytesample can be compared to a catalog of a sample from the same type oforganism as the first analyte sample, a sample from the same type oftissue as the first analyte sample, a sample from the same organism asthe first analyte sample, a sample obtained from the same source but attime different from that of the first analyte sample, a sample from anorganism different from that of the first analyte sample, a sample froma type of tissue different from that of the first analyte sample, asample from a strain of organism different from that of the firstanalyte sample, a sample from a species of organism different from thatof the first analyte sample, or a sample from a type of organismdifferent from that of the first analyte sample.

The same type of tissue is tissue of the same type such as liver tissue,muscle tissue, or skin (which may be from the same or a differentorganism or type of organism). The same organism refers to the sameindividual, animal, or cell. For example, two samples taken from apatient are from the same organism. The same source is similar butbroader, referring to samples from, for example, the same organism, thesame tissue from the same organism, the same analyte, or the sameanalyte sample. Samples from the same source that are to be compared canbe collected at different times (thus allowing for potential changesover time to be detected). This is especially useful when the effect ofa treatment or change in condition is to be assessed. Samples from thesame source that have undergone different treatments can also becollected and compared using the disclosed method. A different organismrefers to a different individual organism, such as a different patient,a different individual animal. Different organism includes a differentorganism of the same type or organisms of different types. A differenttype of organism refers to organisms of different types such as a dogand cat, a human and a mouse, or E. coli and Salmonella. A differenttype of tissue refers to tissues of different types such as liver andkidney, or skin and brain. A different strain or species of organismrefers to organisms differing in their species or strain designation asthose terms are understood in the art.

When comparing catalogs of analytes obtained from related samples, it ispossible to identify the presence of a subset of correlated pairs oflabeled analytes and their altered forms. The disclosed method can beused to detect the original labeled analytes (and determinecharacteristics of them) and the altered form of the labeled analytes.This pair of detected analytes will be characteristic of the analytethat is labeled and the specific reporter signal used (although notnecessarily unique).

1. Reporter Signal Protein Labeling

One form of reporter signal labeling, referred to as reporter signalprotein labeling, involves detection of proteins by detecting a reportersignal, labeled protein, or both; or by distinguishing differentreporter signals, different labeled proteins, or both. Detection of thereporter signals results in detection of the corresponding labeledproteins. Detection of the labeled proteins results in detection of thecorresponding proteins. Thus, the disclosed method is a generaltechnique for labeling, detection, and quantitation of proteins.

In one embodiment, the disclosed method can involve two basic steps. Afiltering, selection, or separation step to separate labeled proteinsfrom other molecules that may be present, and a detection step thatdetects a reporter signal, labeled protein, or both; or thatdistinguishes different reporter signals, different labeled proteins, orboth. The labeled proteins preferably are distinguished and/or separatedfrom other molecules based on some common property shared by theattached reporter signals but not present in most (or, preferably, all)other molecules present. The labeled proteins can also be distinguishedand/or separated from other molecules based on a common property of thelabeled protein as a whole, such as the mass-to-charge ratio of thelabeled protein. The separated labeled proteins are then treated and/ordetected in such a way that the different reporter signals, differentlabeled proteins, or both, are distinguishable.

The disclosed method can be used in many modes. For example, thedisclosed method can be used to detect a specific protein (in a specificsample or in multiple samples) or multiple proteins (in a single sampleor multiple samples). In each case, the protein(s) to be detected can beseparated either from other, unlabeled proteins or from other moleculeslacking a property of the labeled protein(s) to be detected. Forexample, proteins in a sample can be generally labeled with reportersignals and some proteins can be separated on the basis of some propertyof the proteins. For example, the separated proteins could have acertain mass-to-charge ratio (separation based on mass-to-charge ratiowill select both labeled and unlabeled proteins having the selectedmass-to-charge ratio). As another example, all of the labeled proteinscan be distinguished and/or separated from unlabeled molecules based ona feature of the reporter signal such as an affinity tag. Wheredifferent affinity tags are used, some labeled proteins can bedistinguished and/or separated from others.

Optionally, the selection step can be preceded by fractionation stepwhere a subset of proteins, including the proteins that are, or will be,labeled, are separated from other components in a sample. For example,proteins having an SH2 domain can be separated from other proteins in acell sample prior to the selection step. Such a step, although notnecessary, can improve the selection step by reducing the number ofextraneous molecules present.

A preferred form of the disclosed method involves filtering of isobariclabeled proteins from other molecules based on mass-to-charge ratio,fragmentation of the reporter signals to produce fragments havingdifferent mass-to-charge ratios, and detection of the differentfragments based on their mass-to-charge ratios. The different fragmentswill include the fragment of the reporter signal and the fragmentedlabeled protein (made up of the protein and the remaining part of thereporter signal). Either or both may be detected and will becharacteristic of the initial labeled protein. The method is bestcarried out using a tandem mass spectrometer. In such an instrument theisobaric reporter signals are first filtered, then reporter signals arefragmented (preferably by collision), and the fragments aredistinguished and detected.

The same sample can be analyzed both with and without fragmentation (byoperating with and without collision gas), and the results compared todetect shifts in mass-to-charge ratio. Both the unfragmented andfragmented results should give diagnostic peaks, with the combination ofpeaks both with and without fragmentation confirming the identity of theprotein (and reporter signal) involved. Such distinctions areaccomplished by using appropriate sets of isobaric reporter signals andallows large scale multiplexing in the detection of proteins.

A preferred form of the disclosed method involves detection of labeledproteins in two or more samples in the same assay. This allows simpleand consistent detection of differences between the proteins in thesamples. Differential detection is accomplished by labeling the proteinsin each sample with a different reporter signal. Preferably, thedifferent reporter signals used for the different samples will make upan isobaric set. In this way, the same labeled protein in each samplewill have the same mass-to-charge ratio as that labeled protein in adifferent sample. Upon fragmentation of the reporter signals, however,each of the fragmented labeled proteins in the different samples willhave a different mass-to-charge ratio and thus each can be separatelydetected. All can be detected in the same measurement. This is atremendous advantage in both time and quality of the data. For example,since the samples are assayed in a single run, there is no need tocorrect or normalize the results of different samples assayed indifferent runs. This allows accurate comparisons of the relative amountsof the same protein or peptide in different samples since that aremeasured in the same run. There would be no differences to causeinconsistency between the samples.

A preferred use for this multiple sample mode of the disclosed method isthe analysis of a time series of samples. Such series are useful fordetecting changes in a sample or reaction over time. For example,changes in expressed proteins in a cell culture over time after additionof a test compound can be assessed. In this mode, different time pointsamples are labeled with different reporter signals, preferably makingup an isobaric set. In this way, the same labeled protein for each timepoint will have the same mass-to-charge ratio as that labeled proteinfrom a different time point. Upon fragmentation of the reporter signals,however, each of the fragmented labeled proteins from the different timepoints will have a different mass-to-charge ratio and thus each can beseparately detected.

The disclosed method can also be used to gather and catalog informationabout unknown proteins. This protein discovery mode can easily link thepresence or pattern of proteins with their analysis. For example, asample of labeled proteins can be compared to proteins in one or moreother samples. Proteins that appear in one or some samples but notothers can be analyzed for composition and/or sequence usingconventional techniques. The object proteins will be distinguishablefrom others by virtue of the disclosed labeling, detection, andquantitation. This mode of the disclosed method is especially useful asan aid to functional genomics or proteomics since proteins discovered todiffer between samples can be characterized. This mode of the method ispreferably carried out using mass spectrometry.

The disclosed method increases the sensitivity and accuracy of detectionof proteins of interest. Preferred forms of the disclosed method makeuse of multistage detection systems to increase the resolution of thedetection of molecules having very similar properties. The methodinvolves at least two stages. The first stage is filtration or selectionthat allows passage or selection of labeled proteins (that is, a subsetof the molecules present), based upon intrinsic properties of thereporter signals (and the attached proteins), and discrimination againstall other molecules. The subsequent stage(s) further separate(s) and/ordetect(s) the labeled proteins that were filtered in the first stage. Akey facet of this method is that a multiplexed set of labeled proteinswill be selected by the filter and the attached reporter signalssubsequently will be cleaved, decomposed, reacted, or otherwise modifiedto realize the identities and/or quantities of the fragmented reportersignals and/or the fragmented labeled proteins in further stages. Thereis a correspondence between the reporter signal and the detecteddaughter fragment(s).

In some embodiments, the disclosed method allows a complex sample ofproteins and/or peptides to be quickly and easily cataloged in areproducible manner. Such a catalog can be compared with other,similarly prepared catalogs of other protein samples to allow convenientdetection of differences between the samples. The catalogs, whichincorporate a significant amount of information about the proteinsamples, can serve as fingerprints of the samples which can be used bothfor detection of related protein samples and comparison of proteinsamples. For example, the presence or identity of specific organisms canbe detected by producing a catalog of proteins and/or peptides of thetest organism and comparing the resulting catalog with referencecatalogs prepared from known organisms. Changes and differences inprotein expression patterns can also be detected by preparing catalogsof proteins from different cell samples and comparing the catalogs.Comparison of protein catalogs produced with the disclosed method isfacilitated by the fine resolution that can be provided with, forexample, mass spectrometry.

Each labeled protein processed in the disclosed method will result in asignal based on the characteristics of the labeled protein (for example,the mass-to-charge ratio). A complex protein sample can produce a uniquepattern of signals. It is this pattern that can allow unique catalogingof protein samples and sensitive and powerful comparisons of thepatterns of signals produced from different protein samples.

The presence, amount, presence and amount, or absence of differentlabeled proteins forms a pattern of signals that provides a signature orfingerprint of the proteins, and thus of the protein sample based on thepresence or absence of specific proteins, peptides or protein fragmentsin the sample. For this reason, cataloging of this pattern of signals(that is, the pattern of the presence, amount, presence and amount, orabsence of labeled proteins) is an embodiment of the disclosed methodthat is of particular interest.

Catalogs can be made up of, or be referred to, as, for example, apattern of labeled proteins, a pattern of the presence of labeledproteins, a catalog of labeled proteins, a catalog of proteins in asample, or a catalog of amino acid sequences in a sample. Theinformation in the catalog is preferably in the form of mass-to-chargeinformation, compositional information (that is, the composition ofamino acids) or, more preferably, in the form of amino acid sequences.Catalogs can also contain or be made up of other information derivedfrom the information generated in the disclosed method (for example, theidentity of the proteins detected), and can be combined with informationobtained or generated from any other source. The informational nature ofcatalogs produced using the disclosed method lends itself to combinationand/or analysis using known bioinformatics systems and methods.

Such catalogs of protein samples can be compared to a similar catalogderived from any other sample to detect similarities and differences inthe samples (which is indicative of similarities and differences in theproteins in the samples). For example, a catalog of a first proteinsample can be compared to a catalog of a sample from the same type oforganism as the first protein sample, a sample from the same type oftissue as the first protein sample, a sample from the same organism asthe first protein sample, a sample obtained from the same source but attime different from that of the first protein sample, a sample from anorganism different from that of the first protein sample, a sample froma type of tissue different from that of the first protein sample, asample from a strain of organism different from that of the firstprotein sample, a sample from a species of organism different from thatof the first protein sample, or a sample from a type of organismdifferent from that of the first protein sample.

When comparing catalogs of proteins obtained from related samples, it ispossible to identify the presence of a subset of correlated pairs oflabeled proteins and their altered forms. The disclosed method can beused to detect the original labeled proteins (and determinecharacteristics of them) and the altered form of the labeled proteins.This pair of detected proteins will be characteristic of the proteinthat is labeled and the specific reporter signal used (although notnecessarily unique).

C. Reporter Signal Calibration

In another form of the method, referred to as reporter signalcalibration (RSC), a form of reporter signals referred to as reportersignal calibrators are mixed with analytes or analyte fragments, thereporter signal calibrators and the analytes or analyte fragments arealtered, and the altered forms of the reporter signal calibrators andaltered forms of the analytes or analyte fragments are detected.Reporter signal calibrators are useful as standards for assessing theamount of analytes present. That is, one can add a known amount of areporter signal calibrator in order to assess the amount of analytepresent comparing the amount of altered analyte or analyte fragmentdetected with the amount of altered reporter signal calibrator detectedand calibrating these amounts with the known amount of reporter signalcalibrator added (and thus the predicted amount of altered reportersignal calibrator).

The disclosed reporter signal calibration method generates, with highsensitivity, unique analyte signatures related to the relative abundanceof different analytes in tissue, microorganisms, or any other biologicalsample. The disclosed method allows one, for example, to define thestatus of a cell or tissue by identifying and measuring the relativeconcentrations of a small but highly informative subset of analytes.Such as measurement is known as an analyte signature. Analyte signaturesare useful, for example, in the diagnosis, grading, and staging ofcancer, in drug screening, and in toxicity testing.

In some embodiments, each analyte or analyte fragment can share one ormore common properties with at least one reporter signal calibrator suchthat the reporter signal calibrators and analytes or analyte fragmentshaving the common property can be distinguished and/or separated fromother molecules lacking the common property.

In some embodiments, reporter signal calibrators and analytes andanalyte fragments can be altered such that the altered form of ananalyte or analyte fragment can be distinguished from the altered formof the reporter signal calibrator with which the analyte or analytefragment shares a common property. In some embodiments, the alteredforms of different reporter signal calibrators can be distinguished fromeach other. In some embodiments, the altered forms of different analytesor analyte fragments can be distinguished from each other.

In some embodiments of reporter signal calibration, the analyte oranalyte fragment is not altered and so the altered reporter signalcalibrators and the analytes or analyte fragments are detected. In thiscase, the analyte or analyte fragment can be distinguished from thealtered form of the reporter signal calibrator with which the analyte oranalyte fragment shares a common property.

In some embodiments the analyte or analyte fragment may be a reportersignal or a fragment of a reporter signal. In this case, the reportersignal calibrators can serve as calibrators for the amount of reportersignal detected.

Reporter signal calibration is preferably used in connection withproteins and peptides (as the analytes). This form of reporter signalcalibration is referred to as reporter signal protein calibration.Reporter signal protein calibration is useful, for example, forproducing protein signatures of protein samples. As used herein, aprotein signature is the presence, absence, amount, or presence andamount of a set of proteins or protein surrogates.

In some embodiments of reporter signal protein calibration, the presenceof labile, scissile, or cleavable bonds in the proteins to be detectedcan be exploited. Peptides, proteins, or protein fragments (collectivelyreferred to, for convenience, as protein fragments in the remainingdescription) containing such bonds can be altered by fragmentation atthe bond. In this way, reporter signal calibrators having a commonproperty (such as mass-to-charge ratio) with the protein fragments canbe used and the altered forms of the reporter signal calibrators and thealtered (that is, fragmented) forms of the protein fragments can bedetected and distinguished. In this regard, although the proteinfragments share a common property with their matching reporter signalcalibrators, the altered forms of the reporter signal calibrators andaltered forms of protein fragments can be distinguished (because, forexample, the altered forms have different properties, such as differentmass-to-charge ratios).

The disclosed reporter signal protein calibration method generates, withhigh sensitivity, unique protein signatures related to the relativeabundance of different proteins in tissue, microorganisms, or any otherbiological sample. The disclosed method allows one, for example, todefine the status of a cell or tissue by identifying and measuring therelative concentrations of a small but highly informative subset ofproteins. Such as measurement is known as a protein signature. Proteinsignatures are useful, for example, in the diagnosis, grading, andstaging of cancer, in drug screening, and in toxicity testing.

As an example of reporter signal protein calibration, a protein sampleof interest can be digested with a serine protease, preferably trypsin.The digest generates a complex mixture of protein fragments. Among theseprotein fragments, there will exist a subset (approximately one proteinfragment among every 400) that contains the dipeptide Asp-Pro. Thisdipeptide sequence is uniquely sensitive to fragmentation during massspectrometry, an thus produces a high yield of ions in fragmentationmode. Since the human proteome consists of at least 2,000,000 distincttryptic peptides, the number of protein fragments containing the Asp-Prosequence is of the order of 5,000. Since some of these may exist asphosphopeptides or other modified forms, the number may be even higher.This number is sufficiently high to permit the selection of a subset(perhaps 50 to 100 or so) of fragmentable protein fragments that issuitable for generating a highly informative protein signature. Peptidesthat contain the Asp-Pro dipeptide sequence, peptides that contain aminoacids that are modified by phosphorylation inside the cell, or peptidesthat contain an internal methionine are particularly preferred for usein reporter signal calibration. Alternatively, tryptic peptidesterminating in arginine may be modified by reaction with acetylacetone(pentane-2,4-dione) to increase the frequency of fragment ions (Dikleret al., J Mass Spectrom 32:1337-49 (1997)). Selection of the subsets ofprotein fragments can be performed using bioinformatics in order tomaximize the information content of the protein signatures.

For this form of reporter signal calibration, the protein digest can bemixed with a specially designed set of reporter signal calibrators, andthen can be analyzed using tandem mass spectrometry. In the case of atandem in space instrument (for example, Q-Tof™ from Micromass), usingfirst quadrupole settings for single-ion filtering (defined by themolecular mass of each unique fragment, which can be obtained fromsequence data), followed by a collision stage for ion fragmentation, andfinally TOF spectrometry of the peptide fragments (generated by cleavageat fragile bonds, such as Asp-Pro, bonds involving a phosphorylatedamino-acid, or bonds adjacent to an oxidized amino-acid such asmethionine sulfoxide, Smith et al., Free Radic Res. 26:103-11 (1997))that arise from the original single-ion. In the second stage, signal tonoise of the TOF measurement is much larger than in a conventional MSexperiment. In general, one reporter signal calibrator can be used foreach protein fragment in the sample that will be used to make up theprotein signature (such protein fragments are referred to as signatureprotein fragments), and each is designed to fragment in an easilydetectable pattern of masses, distinct from the fragment masses of theunfragmented signature protein fragments. The quadrupole filteringsettings are then varied in sequence over a range of values (fifty, forexample), corresponding to the masses of each of the protein fragmentspreviously chosen to comprise the protein signature (that is, thesignature protein fragments). At each filtered mass setting, there willbe two types of signals detectable by TOF after fragmentation, one setderived from the tryptic peptide (that is, the original proteinfragment), and another set corresponding to the reporter signalcalibrator. The reporter signal calibrator permits one to calculaterelative abundance for each of the signature protein fragments. Theserelative abundance ratios, determined for a given sample, constitute theprotein signature. The detection limit of the tandem mass spectrometerin MS/MS mode, is remarkably good, perhaps of the order of 500 moleculesof peptide. This level of detection is approximately 1,000 times betterthan that for MALDI-TOF mass spectrometry, and should permit thegeneration of protein signatures from single cells.

As can be seen, for this form of reporter signal calibration, theavailability of the sequence of the entire human genome, as well as thegenomes of many other organisms, can facilitate the identification ofprotein fragments that are unique in the context of all known proteins.That is, the sequence information can be used to identify peptides thatwill be generated in a protein signature and guide selection of reportersignal calibrators.

D. Reporter Signal Fusions

In another form of the disclosed method, referred to as reporter signalfusions (RSF), reporter signal peptides are joined with a protein orpeptide of interest in a single amino acid segment, and the reportersignal peptide, reporter signal fusion, altered forms of the reportersignal peptide, and/or altered forms of the reporter signal fusion canbe detected.

The disclosed method provides sensitive monitoring and detection of theproteins and peptides to which reporter signal peptides are fused. Inparticular, the method provides sensitive and multiplex detection ofexpression of particular proteins and peptides of interest, and/or ofthe genes, vectors, and expression constructs encoding the proteins andpeptides of interest.

The disclosed method can use cells, cell lines, and organisms that haveparticular protein(s), gene(s), vector(s), and/or expression sequence(s)labeled (that is, associated with or involved in) reporter signalfusions. For example, a set of nucleic acid constructs, each encoding areporter signal fusion with a different reporter signal peptide, can beused to uniquely label a set of cells, cell lines, and/or organisms.Processing, in the disclosed method, of a sample from any of the labeledsources can result in a unique altered form of the reporter signalpeptide (or the amino acid segment or an amino acid subsegment) for eachof the possible sources that can be distinguished from the other alteredforms. Detection of a particular altered form identifies the source fromwhich it came. As a more specific example, a genetically modified plantline (for example, a Roundup resistant corn line) into which a nucleicacid construct encoding a reporter signal fusion has been introduced canbe identified by detecting the reporter signal fusion.

The disclosed method can also be used to assess the state and/orexpression of particular pathways, regulatory cascades, and other suitesof genes, proteins, vectors, and/or expressions sequences. By usingreporter signal fusions to “label” such pathways, cascades, etc. withinthe same cell, cell line, or organism, or across a set of cells, celllines, or organisms, the pathways, cascades and other systems can beassessed in a single assay and/or compared across cells, cell lines, ororganisms. For example, nucleic acid segments encoding reporter signalfusions can be associated with endogenous expression sequences ofinterest, endogenous genes of interest, exogenous expression sequencesof interest, exogenous genes of interest, or a combination, and theexpression of the genes and/or expression sequences assessed bydetecting the reporter signal peptides and/or reporter signal fusions.Many other modes of the method are possible, a number of which aredescribed in the illustrations below. In particular, the disclosedreporter signal fusions can be used in the disclosed method for purposesanalogous to any purpose that green fluorescent protein and greenfluorescent protein fusions are used. However, the disclosed method canmake use of reporter signal fusions in many more ways and for moreuseful purposes than green fluorescent protein at least because of theability to multiplex the disclosed reporter signal fusions.

Nucleic acid sequences encoding reporter signal peptides can beengineered into particular exons of a gene. This would be the normalsituation when the gene encoding the protein to be fused containsintrons, although sequence encoding a reporter signal peptide can besplit between different exons to be spliced together. Placement ofnucleic acid sequences encoding reporter signal peptides into particularexons is useful for monitoring and analyzing alternative splicing ofRNA. The appearance of a reporter signal peptide in the final proteinindicates that the exon encoding the reporter signal peptide was splicedinto the mRNA.

The disclosed method can provide sensitive detection of one or multipleproteins (that is, proteins to which reporter signal peptides arefused). In the method, proteins fused with reporter signal peptides areanalyzed using the reporter signal peptides to distinguish the reportersignal fusions. Detection of the reporter signal peptides indicates thepresence of the corresponding protein(s). The detected protein(s) canthen be analyzed using known techniques. The reporter signal fusionsprovide a unique protein/label composition that can specificallyidentify the protein(s). This is accomplished through the use of thespecialized reporter signal peptides as the labels.

In the method, reporter signal fusions can be fragmented, such as byprotease digestion, prior to analysis. An expression sample to beanalyzed can also be subjected to fractionation or separation to reducethe complexity of the samples. Fragmentation and fractionation can alsobe used together in the same assay. Such fragmentation and fractionationcan simplify and extend the analysis of the expression.

In the method, reporter signal peptides can be fragmented, decomposed,reacted, derivatized, or otherwise modified, preferably in acharacteristic way. This allows a protein to which the reporter signalpeptide is fused to be identified by detection of one or more of theproducts of the reporter signal fusion following fragmentation,decomposition, reaction, derivatization, or other modification of thereporter signal peptide. The protein can also be identified by thecorrelated detection of the reporter signal fusion and one or more ofthe products of the reporter signal fusion following fragmentation,decomposition, reaction, derivatization, or other modification of thereporter signal peptide. The alteration of the reporter signal peptidewill alter the reporter signal fusion in a characteristic and detectableway. Together, the detection of a characteristic reporter signal fusionand a characteristic product of (that is, altered form of) the reportersignal fusion can uniquely identify the protein (although the alteredform alone can be detected, if desired). In this way, using thedisclosed method, expression of one or more proteins can be detected,either alone or together (for example, in a multiplex assay). Further,expression of one or more proteins in one or more samples can bedetected in a multiplex manner. Preferably, for mass spectrometryreporter signals, the reporter signal peptides are fragmented to yieldfragments of similar charge but different mass.

Preferably, the reporter signal peptides are fragmented to yieldfragments of similar charge but different mass. This allows eachreporter signal fusion (and/or each reporter signal peptide) in a set tobe distinguished by the different mass-to-charge ratios of the fragmentsof (that is, altered forms of) the reporter signal peptides. This ispossible since the fragments of the different reporter signal peptides(or the fragments of the reporter signal fusions) can be designed tohave different mass-to-charge ratios. In the disclosed method, thisallows each reporter signal fusion to be distinguished by themass-to-charge ratios of the reporter signal fusions after fragmentationof the reporter signal peptide.

Alteration of reporter signals peptides in reporter signal fusions canproduce a variety of altered compositions. Any or all of these alteredforms can be detected. For example, the altered form of the reportersignal peptide can be detected, the altered form of the amino acidsegment (which contains the reporter signal peptide) can be detected,the altered form of a subsegment of the amino acid segment can bedetected, or a combination of these can be detected. Where the reportersignal peptide is altered by fragmentation, the result generally will bea fragment of the reporter signal peptide and an altered form of theamino acid segment containing the protein or peptide of interest and aportion of the reporter signal peptide (that is, the portion not in thereporter signal peptide fragment).

The protein or peptide of interest also can be fragmented. The resultwould be a subsegment of the amino acid segment. The amino acidsubsegment would contain the reporter signal peptide and a portion ofthe protein or peptide of interest. When the reporter signal peptide inan amino acid subsegment is altered (which can occur before, during, orafter fragmentation of the amino acid segment), the result is an alteredform of the amino acid subsegment (and an altered form of the reportersignal peptide). This altered form of amino acid subsegment can bedetected. Where the reporter signal peptide is altered by fragmentation,the result generally will be a fragment of the reporter signal peptideand an altered form of (that is, fragment of) the amino acid subsegment.In this case, the altered form of the amino acid subsegment will containa portion of the protein or peptide of interest and a portion of thereporter signal peptide (that is, the portion not in the reporter signalpeptide fragment).

Cells, cell lines, organisms, and expression of genes and proteins canbe detected using the disclosed reporter signal fusions in a variety ofways. For example, the protein and attached reporter signal peptide canbe detected together, one or more peptides of the protein and theattached reporter signal peptide(s) can be detected together, thefragments of the reporter signal peptide can be detected, or acombination. Preferred detection involves detection of the reportersignal fusion both before and after fragmentation of the reporter signalpeptide.

A preferred form of the disclosed method involves correlated detectionof the reporter signal peptides both before and after fragmentation ofthe reporter signal peptide. This allows genes, proteins, vectors, andexpression constructs “labeled” with a reporter signal peptide to bedetected and identified via the change in the reporter signal fusionand/or reporter signal peptide. That is, the nature of the reportersignal fusion or reporter signal peptide detected (non-fragmented versusfragmented) identifies the gene, protein, vector, or nucleic acidconstruct from which it was derived. Where the reporter signal fusionsand reporter signal peptides are detected by mass-to-charge ratio, thechange in mass-to-charge ratio between fragmented and non-fragmentedsamples provides the basis for comparison. Such mass-to-charge ratiodetection is preferably accomplished with mass spectrometry.

As an example, a fusion between a protein of interest and a reportersignal peptide designed as a mass spectrometry label can be expressed.The reporter signal fusion can be subjected to tryptic digest followedby mass spectrometry of the resulting materials. A peak corresponding tothe tryptic fragment containing the reporter signal peptide will bedetected. Fragmentation of the reporter signal peptide in a collisioncell in the mass spectrometer would result in a shift in the peakcorresponding to the loss of a portion of the attached reporter signalpeptide, the appearance of a peak corresponding to the lost fragment, ora combination of both events. Significantly, the shift observed willdepend on which reporter signal peptide is fused to the protein sincedifferent reporter signal peptides will, by design, produce fragmentswith different mass-to-charge ratios. The combination event of detectionof the parent mass-to-charge (with no collision gas) and themass-to-charge corresponding to the loss of the fragment from thereporter signal peptide (with collision gas) indicates a reporter signalfusion (thus indicating expression of the reporter signal fusion and ofthe gene, vector, or construct encoding it).

A powerful form of the disclosed method is use of reporter signalfusions to assay multiple samples (for example, time series assays orother comparative analyses). Knowledge of the temporal response of abiological system following perturbation is a very powerful process inthe pursuit of understanding the system. To follow the temporal responsea sample of the system is obtained (for example, cells from a cellculture, mice initially synchronized and sacrificed) at determined timesfollowing the perturbation. Knowledge of spatial protein profiles (forexample, relative position within a tissue section) is a very powerfulprocess in the pursuit of understanding the biological system.

The reporter signal fusions are preferably detected using massspectrometry which allows sensitive distinctions between molecules basedon their mass-to-charge ratios. A set of isobaric reporter signalpeptides or reporter signal fusions can be used for multiplex labelingand/or detection of the expression of many genes, proteins, vectors,expression constructs, cells, cell lines, and organisms since thereporter signal peptide fragments can be designed to have a large rangeof masses, with each mass individually distinguishable upon detection.Where the same gene, protein, vectors, expression construct, cell, cellline, or organism (or the same type of gene, protein, vector, expressionconstruct, cell, cell line, or organism) is labeled with a set ofreporter signal fusions that are isobaric or that include isobaricreporter signal peptides (by, for example, “labeling” the same gene,protein, vector, expression construct, cell, cell line, or organism indifferent samples), the set of reporter signal fusions or reportersignal peptides that results will also be isobaric. Fragmentation of thereporter signal peptides will split the set of reporter signal peptidesinto individually detectable reporter signal fusion fragments andreporter signal peptide fragments of characteristically different mass.

A preferred form of the disclosed method involves filtering of isobaricreporter signal fusions or reporter signal peptides from other moleculesbased on mass-to-charge ratio, fragmentation of the reporter signalpeptides to produce fragments having different masses, and detection ofthe different fragments based on their mass-to-charge ratios. The methodis best carried out using a tandem mass spectrometer as describedelsewhere herein.

Nucleic acid sequences and segments encoding reporter signal fusions canbe expressed in any suitable manner. For example, the disclosed nucleicacid sequences and nucleic acid segments can be expressed in vitro, incells, and/or in cells in organism. Many techniques and systems forexpression of nucleic acid sequences and proteins are known and can beused with the disclosed reporter signal fusions. For example, manyexpression sequences, vector systems, transformation and transfectiontechniques, and transgenic organism production methods are known and canbe used with the disclosed reporter signal peptide method andcompositions.

For example, kits for the in vitro transcription/translation of DNAconstructs containing promoters and nucleic acid sequence to betranscribed and translated are known (for example, PROTEINscript-PRO™from Ambion, Inc. Austin Tex.; Wilkinson (1999) “Cell-Free And Happy: InVitro Translation And Transcription/Translation Systems”, The Scientist13[13]:15, Jun. 21, 1999). Such constructs can be used in the genomicDNA of an organism, in a plasmid or other vector that may be transfectedinto an organism, or in in vitro systems. For example, constructscontaining a promoter sequence and a nucleic acid sequence that,following transcription and translation, results in production of greenfluorescence protein or luciferase as a reporter/marker in in vivosystems are known (for example, Sawin and Nurse, “Identification offission yeast nuclear markers using random polypeptide fusions withgreen fluorescent protein.” Proc Natl Acad Sci USA 93(26):15146-51(1996); Chatterjee et al., “In vivo analysis of nuclear protein trafficin mammalian cells.” Exp Cell Res 236(1):346-50 (1997); Patterson etal., “Quantitative imaging of TATA-binding protein in living yeastcells.” Yeast 14(9):813-25 (1998); Dhandayuthapani et al., “Greenfluorescent protein as a marker for gene expression and cell biology ofmycobacterial interactions with macrophages.” Mol Microbiol 17(5):901-12(1995); Kremer et al., “Green fluorescent protein as a new expressionmarker in mycobacteria.” Mol Microbiol 17(5):913-22 (1995); Reilander etal., “Functional expression of the Aequorea victoria green fluorescentprotein in insect cells using the baculovirus expression system.”Biochem Biophys Res Commun 219(1):14-20 (1996); Mankertz et al.,“Expression from the human occludin promoter is affected by tumornecrosis factor alpha and interferon gamma” J Cell Sci, 113:2085-90(2000); White et al., “Real-time analysis of the transcriptionalregulation of HIV and hCMV promoters in single mammalian cells” J CellSci, 108:441-55 (1995)). Green fluorescence protein, or variants, havebeen shown to be stably incorporated and not interfere with theorganism—generally GFP is larger relative to the disclosed reportersignal peptides (GFP from Aequorea Victoria is 238 amino acids in size;NCBI GI:606384), and thus the reporter signal peptides are less likelyto disrupt an expression system to which they are added.

Techniques are known for modifying promoter regions such that theendogenous promoter is replaced with a promoter-reporter construct, forexample, where the reporter is green fluorescent protein (Patterson etal., “Quantitative imaging of TATA-binding protein in living yeastcells.” Yeast 14(9): 813-25 (1998)) or luciferase. Transcription factorconcentrations are followed by monitoring the GFP or luciferase. Thesetechniques can be used with the disclosed reporter signal fusions andreporter signal fusion constructs. Techniques are also known fortargeted knock-in of nucleic acid sequences into a gene of interest,typically under control of the endogenous promoter. Such techniques,which can be used with the disclosed method and compositions, have beenused to introduce reporter/markers of the transcription and translationof the gene where then nucleic acid was inserted. The same techniquescan be used to place the disclosed reporter signal fusions under controlof endogenous expression sequences. Alternately, non-targeted knock-ins(techniques for which are also known) can be used to follow the level oractivity of transcription factors.

As with reporter signals generally, reporter signal peptides can be usedin sets where the reporter signal peptides in a set can have one or morecommon properties that allow the reporter signal peptides to beseparated or distinguished from molecules lacking the common property.In the case of reporter signal fusions, amino acid segments and aminoacid subsegments can be used in sets where the amino acid segments andamino acid subsegments in a set can have one or more common propertiesthat allow the amino acid segments and amino acid subsegments,respectively, to be separated or distinguished from molecules lackingthe common property. In general, the component(s) of the reporter signalfusions having common properties can depend on the component(s) to bedetected and/or the mode of the method being used.

Nucleic acid molecules encoding reporter signal fusions can be used insets where the reporter signal peptides in the reporter signal fusionsencoded by a set of nucleic acid molecules can have one or more commonproperties that allow the reporter signal peptides to be separated ordistinguished from molecules lacking the common property. Similarly,nucleic acid molecules encoding amino acid segments can be used in setswhere the reporter signal peptides in the amino acid segments encoded bya set of nucleic acid molecules can have one or more common propertiesthat allow the reporter signal peptides to be separated or distinguishedfrom molecules lacking the common property. Nucleic acid moleculesencoding amino acid segments can be used in sets where the amino acidsegments encoded by a set of nucleic acid molecules can have one or morecommon properties that allow the amino acid segments to be separated ordistinguished from molecules lacking the common property.

Nucleic acid segments (which, generally, are part of nucleic acidmolecules) encoding reporter signal fusions can be used in sets wherethe reporter signal peptides in the reporter signal fusions encoded by aset of nucleic acid segments can have one or more common properties thatallow the reporter signal peptides to be separated or distinguished frommolecules lacking the common property. Similarly, nucleic acid segmentsencoding amino acid segments can be used in sets where the reportersignal peptides in the amino acid segments encoded by a set of nucleicacid molecules can have one or more common properties that allow thereporter signal peptides to be separated or distinguished from moleculeslacking the common property. Nucleic acid segments encoding amino acidsegments can be used in sets where the amino acid segments encoded by aset of nucleic acid molecules can have one or more common propertiesthat allow the amino acid segments to be separated or distinguished frommolecules lacking the common property. Other relationships betweenmembers of the sets of nucleic acid molecules, nucleic acid segments,amino acid segments, reporter signal peptides, and proteins of interestare contemplated.

Reporter signal fusions can include other components besides a proteinof interest and a reporter signal peptide. For example, reporter signalfusions can include epitope tags or flag peptides. Epitope tags and flagpeptides can serve as tags by which reporter signal fusions can beseparated, distinguished, associated, and/or bound. The use of epitopetags and flag peptides generally is known and can be adapted for use inthe disclosed reporter signal fusions.

In preferred embodiments, reporter signal peptides, reporter signalfusions (or amino acid segments), nucleic acid segments encodingreporter signal fusion, and/or nucleic acid molecules comprising nucleicacid segments encoding reporter signal fusions are used in sets wherethe reporter signal peptides, the reporter signal fusions, and/orsubsegments of the reporter signal fusions constituting or present inthe set have similar properties (such as similar mass-to-charge ratios).The similar properties allow the reporter signals, the reporter signalfusions, or subsegments of the reporter signal fusions to bedistinguished and/or separated from other molecules lacking one or moreof the properties. Preferably, the reporter signals, the reporter signalfusions, or subsegments of the reporter signal fusions constituting orpresent in a set have the same mass-to-charge ratio (m/z). That is, thereporter signals, the reporter signal fusions, or subsegments of thereporter signal fusions in a set are isobaric. This allows the reportersignals, the reporter signal fusions, or subsegments of the reportersignal fusions to be separated precisely from other molecules based onmass-to-charge ratio. The result of the filtering is a huge increase inthe signal to noise ratio (S/N) for the system, allowing more sensitiveand accurate detection.

Preferred reporter signal peptides for use in reporter signal fusionsused in or associated with different genes, proteins, vectors,constructs, cells, cell lines, or organisms would be those usingdifferentially distributed mass. In particular, the use of alternativeamino acid sequences using the same amino acid composition is preferred.

Although reference is made above and elsewhere herein to detection of,and fusion with, a “protein” or “proteins,” the disclosed method caninvolve proteins, peptides, and fragments of proteins or peptides. Thus,reference to a protein herein is intended to refer to proteins,peptides, and fragments of proteins or peptides unless the contextclearly indicates otherwise. As used herein “reporter signal fusion”refers to a protein, peptide, or fragment of a protein or peptide towhich a reporter signal peptide is fused (that is, joined by peptidebond(s) in the same polypeptide chain) unless the context clearlyindicates otherwise. The reporter signal peptide(s) can be fused to aprotein in any arrangement, such as at the N-terminal end of theprotein, at the C-terminal end of the protein, in or at domainjunctions, or at any other appropriate location in the protein. In someforms of the method, it is desirable that the protein remain functional.In such cases, terminal fusions or inter-domain fusions are preferably.Those of skill in the art of protein fusions generally know how todesign fusions where the protein of interest remains functional. Inother embodiments, it is not necessary that the protein remainfunctional in which case the reporter signal peptide and protein canhave any desired structural organization.

The reporter signal fusions can be produced by expression from nucleicacid molecules encoding the fusions. Thus, the disclosed fusionsgenerally can be designed by designing nucleic acid segments that encodeamino acid segments where the amino acid segments comprise a reportersignal peptide and a protein or peptide of interest. A given nucleicacid molecule can comprise one or more nucleic acid segments. A givennucleic acid segment can encode one or more amino acid segments. A givenamino acid segment can include one or more reporter signal peptides andone or more proteins or peptides of interest. The disclosed amino acidsegments consist of a single, contiguous polypeptide chain. Thus,although multiple amino acid segments can be part of the same contiguouspolypeptide chain, all of the components (that is, the reporter signalpeptide(s) and protein(s) and peptide(s) of interest) of a given aminoacid segment are part of the same contiguous polypeptide chain.

Reporter signal fusions can be used to monitor and analyze alternativeRNA splicing. A central problem in translating the information in thegenome to protein expression is an understanding of mRNA alternativeprocessing, and the generation of protein isoforms via alternative exonutilization (Black, “Protein diversity from alternative splicing: achallenge for bioinformatics and post-genome biology” Cell 103:367-70(2000)). Many examples of the use of alternative pre-mRNA splicing togenerate protein isoform diversity exist, such as in the control oferythroid differentiation (see, for example, Hou and Conboy, “Regulationof alternative pre-mRNA splicing during erythroid differentiation” CurrOpin Hematol 8:74-9 (2001)). Often the detection of complex,alternatively spliced protein isoforms is a difficult task, since exonsmay be as small as 6 amino acids in protein of over 2000 amino acids(see, for example, Cianci et al., “Brain and muscle express a uniquealternative transcript of all spectrin” Biochem 38:15721-15730 (1999)).

Exon utilization and processing information can be obtained by insertionof a nucleic acid sequence encoding a reporter signal into the exonsequence of interest (thus forming a nucleic acid segment that encodes areporter signal fusion). The insertions can be made, for example, intogenomic DNA, appropriate mini-gene constructs, or non-endogenouspre-mRNA introduced into the cell. Use of a set of reporter signalsallows the multiplexed readout of all exons of a translated protein atone time. The use of mini-gene constructs or constructs incorporatingshort exogenous open-reading frame DNA sequences into exons, and theincorporation of foreign DNA in association with functional intronsplice elements are developed technologies that can be used forincorporation of reporter signals (see, for example, Gee et al.,“Alternative splicing of protein 4.1R exon 16: ordered excision offlanking introns ensures proper splice site choice” Blood 95:692-9(2000); Kikumori et al., “Promiscuity of pre-mRNA spliceosome-mediatedtrans splicing: a problem for gene therapy?” Hum Gene Ther 12:1429-41(2001); Malik et al., “Effects of a second intron on recombinant MFGretroviral vector” Arch Virol 146:601-9 (2001); Virts and Raschke, “Therole of intron sequences in high level expression from CD45 cDNAconstructs” J Biol Chem 276:19913-20 (2001)). Detection of the reportersignals, the amounts of the reporter signals, and the knowledge of whichreporter signal correlates with which exon, provides information aboutexon usage and alternative splicing.

The disclosed reporter signal fusions also can be used in the detectionand analysis of protein interactions with other proteins and molecules.For example interaction traps for protein-protein interactions includethe well known yeast two-hybrid (Fields and Song. “A novel geneticsystem to detect protein-protein interactions” Nature 340:245-6 (1989);Uetz et al., “A comprehensive analysis of protein-protein interactionsin Saccharomyces cerevisiae” Nature 403:623-7 (2000)) and relatedsystems (Ausubel et al., Current Protocols in Molecular Biology, JohnWiley & Sons, Inc., 2001; Van Criekinge and Beyaert, “Yeast two-hybrid:state of the art” Biological Procedures Online, 2(1), 1999).Incorporation of nucleic acid sequence encoding a peptide reportersignal can be introduced into these systems, for example at a terminusof the ordinarily used LacZ selection region (LacZ selection isdescribed in, for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, second edition, 1989, Cold Spring Harbor LaboratoryPress, New York). A set of such incorporated sequences (for example, ina set of such plasmids, where each plasmid has a reporter signal codingsequence and the LacZ functionality), allows the unambiguous detectionof many interactions simultaneously rather (as many differentinteractions as reporter signals used).

In another mode of reporter signal fusions, a nucleic acid sequenceencoding a reporter signal could be added to sequence encoding theconstant (C) region of T cell and B cell receptors. The reporter signalwould appear in T or B cell receptors when that C region is spliced to aJ region following transcription.

In another mode of reporter signal fusions, referred to as reportersignal presentation, the presentation of specific antigenic peptides bymajor histocompatibility (MHC) and non-major histocompatibilitymolecules can be detected and analyzed. It is well known that proteinantigens are processed by antigen presenting cells and that smallpeptides, typically 8-12 amino acids are presented by Class I and ClassII MHC molecules for recognition by T cells. The study of specific Tcell/peptide-MHC complexes is technically challenging due variouslabeling requirements (either radioactive or fluorescence) and thecommon reliance on antibody reagents that recognize specific receptorsand/or peptide-MHC complexes.

There is a need to be able to further expand our knowledge of antigenprocessing and antigen presentation. Reporter signals that have beenengineered into specific protein antigens could provide novel insightinto this process and enable new experimental approaches. For instance,consider two viral or bacterial proteins, protein A and protein B, thatdiffer by only a few amino acids. It would be useful to know if they areprocessed and presented to immune cells (for example, T cells) with thesame efficiency. By engineering reporter signals into protein A andengineered protein B to antigen presenting cells, one could test for thepresence of the different reporter signals presented on and thusdetermine if the proteins are efficiently processed and presented. Thepresence of reporter signal A (present in protein A) but not reportersignal B (present in protein B), indicates that protein A is processedand that protein B is not. The lack of antigen processing of protein Bmay then be an explanation of why a virus or bacteria escapes immunesurveillance by the immune system. Antigenic peptides are characterizedby conserved anchor residues near both the amino and carboxy ends, withmore heterogeneity tolerated in the middle. This middle heterogeneity isthus a preferred site for addition of a reporter signal peptide.

E. Rearranging Reporter Signals

Another embodiment of the disclosed method and compositions, referred toas rearranging reporter signals, enables one to detect the occurrence ofspecific gene rearrangement events, their protein products, and specificcell populations bearing those receptors. Rearranging reporter signalswill also allow one to follow the progression or development of certainreceptors and cells or populations of cells by monitoring the presenceand/or absence of a reporter signal. Design considerations forrearranged reporter signals are analogous to those required for reportersignal fusions as described elsewhere herein.

Most embodiments of the disclosed method involve intact reporter signalsthat are associated with analytes in various ways. Rearranging reportersignals make use of processes, such as biological processes, to formreporter signals by specific rearrangement of the reporter signal piecesor rearrangement of nucleic acid segments encoding only portions ofreporter signals. One form of rearranging reporter signals utilizesendogenous biological systems, such as the variable-diversity-joining(V-D-J) gene rearrangement machinery present in the mammalian immunesystem. In this system, short stretches of germline DNA (the V, D & Jgene fragments) that are not contiguous, are brought together(recombined) prior to serving as a template for transcription. Generearrangement occurs in white blood cells such as T and B lymphocytesand is a key mechanism for generating diversity of T cell and B cellantigen receptors. Theoretically, billions of different receptors can begenerated. This level of complexity makes it difficult to detect thepresence of rare rearrangement events, or receptors. PCR based assaysand flow cytometry approaches are now used to study receptor diversity.However, PCR approaches are laborious and do not provide any informationon the status of expressed protein. Flow cytometry approaches havelimited multiplexing capabilities due to emission spectra overlap of thefluorescent probes used.

If one desired to test for 50-100 T cell or B cell receptors, one wouldneed to make use of a similar number of antibodies to those receptors,something that in practice is not done. Therefore, there is a real needfor methods that would allow highly sensitive and specific detection ofspecific receptors in a highly complex pool of receptors. The ability tohighly multiplex this approach would enable currently unattainableexperimental approaches. The disclosed reporter signal technology allowslarge scale multiplexing of signals for detection.

As an example of rearranging reporter signals, transgenic mice can begenerated in which nucleic acid sequences encoding reporter signals havebeen engineered into the mouse germline. Methods for doing this are wellknown in the art and include using standard molecular biology methods toengineer rearranging reporter signal into, for example, yeast orbacterial artificial chromosomes (YACs or BACs) and then using theseconstructs to generate transgenic mice.

As an example of the use of immunoglobulin rearrangement for rearrangingreporter signals, part of a reporter signal could be encoded on the Dregion and another part of the reporter signal could be encoded on the Jregion. Upon a rearrangement event that joined the D and J regionsencoding these “partial” reporter signals, a coding sequence for a“complete” reporter signal would be generated. Following transcriptionand translation, the reporter signal would be encoded within the proteinproduct. The reporter signal could then be detected as describedelsewhere herein. In the absence of a rearrangement event that joins theengineered D and J region, no reporter signal would be detected. Byincluding sequences encoding parts of a variety of reporter signals withdifferent D and J regions, a variety of different reporter signals canbe generated by rearrangement, a different, and diagnostic, reportersignal for each of the different possible rearrangements. This systemalso could be extended to include, for example, reporter signals splitamong three or more gene regions (for example, V-D-J, V-D-D-J, etc) withthe result that multiple rearrangement events would produce the reportersignal. In this mode, the combinations of rearrangements of the reportersignal parts can give rise to an large number of different reportersignals, each characterized by the specific reporter signal partsrearranged to form the reporter signal.

Transgenic mice carrying rearranging reporter signals would enable oneto address questions that would otherwise be very difficult orimpossible to address. For instance, one could dissect what specific Tand B cell receptors (out of the thousands or millions possible) respondto specific stimuli or what cell types are present at certain stages ofdevelopment.

F. Lipid Reporter Signals

The disclosed method and compositions also can be used to monitor lipidcomposition, distribution, and processing. Lipids are hydrophobicbiomolecules that have high solubility in organic solvents. They have avariety of biological roles that make them valuable targets formonitoring. As a nutritional source, lipids (together withcarbohydrates) constitute an important source of cellular energy andmetabolic intermediates needed for cell signaling and other processes.Lipids processed for energy conversion typically pass through a varietyof enzymatic pathways, generating many intermediates. A summary of thesecycles is available in most modern biochemistry texts (see, for example,Stryer, 1995). Monitoring the processing of acyl chain intermediates asthey are metabolized is an important tool in lipid and cell biologicalresearch, as well as for the clinical detection of biochemical diseasessuch as medium-chain acyl-CoA dehydrogenase deficiencies (see, forexample, Zschocke et al., “Molecular and functional characterization ofmild MCAD deficiency.”, Hum Genet 108:404-8 (2001)). Incorporatingreporter signals into, or associating reporter signals with, lipids canimprove methods of detecting lipids (such as Andresen et al.,“Medium-chain acyl-CoA dehydrogenase (MCAD) mutations identified byMS/MS-based prospective screening of newborns differ from those observedin patients with clinical symptoms: identification and characterizationof a new, prevalent mutation that results in mild MCAD deficiency” Am JHum Genet 68:1408-18. (2001)) by allowing, for example, more rapid andmultiplex detection of processed acyl chain intermediates.

In another role, lipids function as the most fundamental and definingcomponent of all biological membranes. The three major types of membranelipids are phospholipids, glycolipids, and cholesterol. The mostabundant of these are the phospholipids, derived either from glycerol orsphingosine. Those based on glycerol typically contain two esterifiedlong-chain fatty acids (14 to 24 carbons) and a phosphorylated alcoholor sugar. Phospholipids based on sphingosine contain a single fattyacid. Collectively these lipids contribute to the structure and fluidityof biological membranes. Cyclic changes in their processing,particularly of acidic glycophosolipids such as phosphatidyl inositol4,5 phosphate, also regulate a wide variety of cellular processes (see,for example, Cantrell, “Phosphoinositide 3-kinase signaling pathways” JCell Sci 114:1439-45 (2001); Payrastre et al., “Phosphoinositides: keyplayers in cell signaling, in time and space” Cell Signal 13:377-87(2001)). Thus, by incorporating reporter signals into, or associatingreporter signals with, the acyl chains of such molecules, the subsequentincorporation of such reporter molecules into either in vitro assayssuch as those used for enzyme determinations or in vivo assays, allowsone to rapidly follow the segregation of these lipids into distinctcellular compartments (for example, golgi versus plasma membrane (see,for example, Godi et al., “ARF mediates recruitment of PtdIns-4-OHkinase-beta and stimulates synthesis of PtdIns(4,5)P2 on the Golgicomplex” Nat Cell Biol 1:280-7 (1999)), and their processing viametabolic and signaling pathways such as those cited above.

It is known that exogenous lipid labels can be incorporated readily intobiological systems, and the disclosed reporter signals also can beincorporated into such systems. For example, spin-labeled acyl fattyacids and phospholipids have been incorporated into the membranes ofphospholipid vesicles and cells (see, for example, Kornberg andMcConnell, “Inside-outside transitions of phospholipids in vesiclemembranes” Biochemistry 10:1111-20 (1971); Komberg and McConnell,“Lateral diffusion of phospholipids in a vesicle membrane” Proc NatlAcad Sci USA 68:2564-8 (1971); Arora et al., “Selectivity oflipid-protein interactions with trypsinized Na, K-ATPase studied byspin-label EPR” Biochim Biophys Acta 1371:163-7 (1998); Alonso et al.,“Lipid chain dynamics in stratum corneum studied by spin label electronparamagnetic resonance” Chem Phys Lipids 104:101-11 (2000)).

Triglycerides, or the acyl chain of sphinoglipids or glycolipids, andcholesterol, may be synthesized to include a reporter signal. An exampleof such a reporter signal would be a lipid made from an aliphatic chainwith a carboxylic acid with a photocleavable bond. Examples ofphotocleavable bonds are described by Glatthar and Geise, Org. Lett,2:2315-2317 (2000); Guillier et al., Chem. Rev. 100:2091-2157 (2000);Wierenga, U.S. Pat. No. 4,086,254; and elsewhere here. A set of reportersignals may be prepared by locating the cleavable bond at differentlocations within an aliphatic chain (thus resulting in fragments ofdifferent mass when the bond is cleaved). The aliphatic chain with aphotocleavable bond constitutes the reporter signal. Such syntheticreporter molecules can be incorporated into synthetic triglycerides by,for example, a dehydration reaction. Once formed, a set of thesesynthetic triglycerides can be introduced into biological systems ofinterest, such as those described above. Reporter signals can berecovered from the biological system of interest for detection andquantitation by, for example, extraction of the lipid into chloroformand release of reporter signals from the trigyceride using a lipase orhydrolysis reaction.

Forms and Embodiments of the Disclosed Methods

A. Reporter Molecule Labeling

Disclosed are methods comprising (a) separating a set of reportersignals, where each reporter signal has a common property, frommolecules lacking the common property, (b) altering the reportersignals, (c) detecting and distinguishing the altered forms the reportersignals from each other.

Also disclosed are methods wherein the common property is mass-to-chargeratio, wherein the reporter signals are altered by altering their mass,wherein the altered forms of the reporter signals are distinguished viadifferences in the mass-to-charge ratio of the altered forms of reportersignals.

Also disclosed are methods wherein the mass of the reporter signals isaltered by fragmentation.

Also disclosed are methods wherein the set of reporter signals comprisestwo or more, three or more, four or more, five or more, six or more,seven or more, eight or more, nine or more, ten or more, twenty or more,thirty or more, forty or more, fifty or more, sixty or more, seventy ormore, eighty or more, ninety or more, or one hundred or more differentreporter signals.

Also disclosed are methods wherein the set of reporter signals comprisesten or more different reporter signals.

Also disclosed are methods wherein the reporter signals are peptides,oligonucleotides, carbohydrates, polymers, oligopeptides, or peptidenucleic acids.

Also disclosed are methods wherein the reporter signals are associatedwith, or coupled to, specific binding molecules, wherein each reportersignal is associated with, or coupled to, a different specific bindingmolecule.

Also disclosed are methods wherein the reporter signals are associatedwith, or coupled to, decoding tags, wherein each reporter signal isassociated with, or coupled to, a different decoding tag.

Also disclosed are methods further comprising, prior to step (a),associating the reporter signals with one or more analytes, wherein eachreporter signal is associated with, or coupled to, a different specificbinding molecule, wherein each specific binding molecule can interactspecifically with a different one of the analytes, wherein the reportersignals are associated with the analytes via interaction of the specificbinding molecules with the analytes.

Also disclosed are methods wherein steps (a) through (c) are repeatedone or more times using a different set of reporter signals each time.

Also disclosed are methods wherein, prior to step (a), the differentsets of reporter signals are associated with different samples.

Also disclosed are methods wherein the different sets of reportersignals each comprise the same reporter signals.

Also disclosed are methods wherein the sets of reporter signals eachcontain a single reporter signal.

Also disclosed are methods wherein not all of the reporter signals inthe set are distinguished and/or separated from molecules lacking thecommon property, not all of the reporter signals are altered, and notall of the altered forms of the reporter signals are detected at thesame time.

Also disclosed are methods wherein all of the reporter signals in theset are distinguished and/or separated from molecules lacking the commonproperty, all of the reporter signals are altered, and all of thealtered forms of the reporter signals are detected at different times.

Also disclosed are methods wherein steps (a) through (c) are performedseparately for each reporter signal.

Also disclosed are methods wherein the reporter signals comprisepeptides, wherein the peptides have the same mass-to-charge ratio andmethods wherein the peptides have the same amino acid composition andmethods wherein the peptides have the same amino acid sequence andmethods wherein each peptide contains a different distribution of heavyisotopes and methods wherein each peptide has a different amino acidsequence and methods wherein each peptide has a labile or scissile bondin a different location.

Disclosed are methods comprising (a) separating one or more reportersignals, where each reporter signal has a common property, frommolecules lacking the common property in each of a plurality of samples,(b) altering the reporter signals, (c) detecting and distinguishing thealtered forms the reporter signals from each other.

Also disclosed are methods wherein the common property is mass-to-chargeratio, wherein the reporter signals are altered by altering their mass,wherein the altered forms of the reporter signals are distinguished viadifferences in the mass-to-charge ratio of the altered forms of reportersignals.

Also disclosed are methods wherein the mass of the reporter signals isaltered by fragmentation.

Also disclosed are methods wherein the reporter signals are associatedwith, or coupled to, specific binding molecules, wherein each reportersignal is associated with, or coupled to, a different specific bindingmolecule.

Also disclosed are methods wherein the reporter signals are associatedwith, or coupled to, decoding tags, wherein each reporter signal isassociated with, or coupled to, a different decoding tag.

Also disclosed are methods further comprising, prior to step (a),associating the reporter signals with one or more analytes, wherein eachreporter signal is associated with, or coupled to, a different specificbinding molecule, wherein each specific binding molecule can interactspecifically with a different one of the analytes, wherein the reportersignals are associated with the analytes via interaction of the specificbinding molecules with the analytes.

Also disclosed are methods wherein steps (a) through (c) are repeatedone or more times using a different set of one or more reporter signalseach time and methods wherein, prior to step (a), the different sets ofreporter signals are associated with different samples and methodswherein the different sets of reporter signals each comprise the samereporter signals and methods wherein the sets of reporter signals eachcontain a single reporter signal.

Also disclosed are methods wherein not all of the reporter signals aredistinguished and/or separated from molecules lacking the commonproperty, not all of the reporter signals are altered, and not all ofthe altered forms of the reporter signals are detected at the same time.

Also disclosed are methods wherein all of the reporter signals aredistinguished and/or separated from molecules lacking the commonproperty, all of the reporter signals are altered, and all of thealtered forms of the reporter signals are detected at different times.

Also disclosed are methods wherein steps (a) through (c) are performedseparately for each sample.

B. Reporter Signal Protein Labeling

Also disclosed are methods comprising (a) separating a set of labeledproteins, wherein each labeled protein comprises a protein or peptideand a reporter signal attached to the protein or peptide, wherein eachreporter signal has a common property, wherein the common propertyallows the labeled proteins comprising the same protein or peptide to bedistinguished and/or separated from molecules lacking the commonproperty, (b) altering the reporter signals, thereby altering thelabeled proteins, (c) detecting and distinguishing the altered forms ofthe labeled proteins from each other.

Also disclosed are methods wherein the common property is mass-to-chargeratio, wherein the reporter signals are altered by altering their mass,wherein the altered forms of the labeled proteins are distinguished viadifferences in the mass-to-charge ratio of the altered forms of thelabeled proteins.

Also disclosed are methods wherein the mass of the reporter signals isaltered by fragmentation.

Also disclosed are methods wherein alteration of the reporter signalsalso alters their charge.

Also disclosed are methods wherein the common property is mass-to-chargeratio, wherein the reporter signals are altered by altering theircharge, wherein the altered forms of the labeled proteins can bedistinguished via differences in the mass-to-charge ratio of the alteredforms of reporter signals.

Also disclosed are methods wherein the set of labeled proteins comprisestwo or more, three or more, four or more, five or more, six or more,seven or more, eight or more, nine or more, ten or more, twenty or more,thirty or more, forty or more, fifty or more, sixty or more, seventy ormore, eighty or more, ninety or more, or one hundred or more differentreporter signals.

Also disclosed are methods wherein the set of labeled proteins comprisesten or more different reporter signals.

Also disclosed are methods wherein the reporter signals are peptides,oligonucleotides, carbohydrates, polymers, oligopeptides, or peptidenucleic acids.

Also disclosed are methods wherein the reporter signals are coupled tothe proteins or peptides.

Also disclosed are methods wherein steps (a) through (c) are performedseparately for each labeled protein.

Also disclosed are methods further comprising, prior to step (a),attaching the reporter signals to one or more proteins, one or morepeptides, or one or more proteins and peptides.

Also disclosed are methods wherein steps are repeated one or more timesusing a different set of reporter signals each time.

Also disclosed are methods wherein, prior to step (a), the differentsets of reporter signals are attached to proteins or peptides indifferent samples.

Also disclosed are methods wherein the different sets of reportersignals each comprise the same reporter signals.

Also disclosed are methods wherein the sets of reporter signals eachcontain a single reporter signal.

Also disclosed are methods wherein not all of the labeled proteins inthe set are distinguished and/or separated from molecules lacking thecommon property, not all of the reporter signals are altered, and notall of the altered forms of the labeled proteins are detected at thesame time.

Also disclosed are methods wherein all of the labeled proteins in theset are distinguished and/or separated from molecules lacking the commonproperty, all of the reporter signals are altered, and all of thealtered forms of the labeled proteins are detected at different times.

Also disclosed are methods wherein steps (a) through (c) are performedseparately for each reporter signal.

Also disclosed are methods wherein the common property is one or moreaffinity tags associated with the reporter signals.

Also disclosed are methods wherein one or more affinity tags areassociated with the reporter signals.

Also disclosed are methods wherein the collection of altered forms ofthe labeled proteins detected constitutes a catalog of proteins.

Also disclosed are methods comprising (a) separating one or more labeledproteins, wherein each labeled protein comprises a protein or peptideand a reporter signal attached to the protein or peptide, wherein eachreporter signal has a common property, wherein the common propertyallows the labeled proteins comprising the same protein or peptide to bedistinguished and/or separated from molecules lacking the commonproperty in each of one or more samples, (b) altering the reportersignals, thereby altering the labeled proteins, (c) detecting anddistinguishing the altered forms the labeled proteins from each other.

Also disclosed are methods wherein the common property is mass-to-chargeratio, wherein the reporter signals are altered by altering their mass,wherein the altered forms of the labeled proteins are distinguished viadifferences in the mass-to-charge ratio of the altered forms of labeledproteins.

Also disclosed are methods wherein the mass of the reporter signals isaltered by fragmentation.

Also disclosed are methods wherein alteration of the reporter signalsalso alters their charge.

Also disclosed are methods wherein the common property is mass-to-chargeratio, wherein the reporter signals are altered by altering theircharge, wherein the altered forms of the labeled proteins can bedistinguished via differences in the mass-to-charge ratio of the alteredforms of reporter signals.

Also disclosed are methods wherein the reporter signals are coupled tothe proteins or peptides.

Also disclosed are methods further comprising, prior to step (a),attaching the reporter signals to one or more proteins, one or morepeptides, or one or more proteins and peptides.

Also disclosed are methods wherein steps are repeated one or more timesusing a different set of one or more reporter signals each time.

Also disclosed are methods wherein, prior to step (a), the differentsets of reporter signals are attached to proteins or peptides indifferent samples.

Also disclosed are methods wherein the different sets of reportersignals each comprise the same reporter signals.

Also disclosed are methods wherein the sets of reporter signals eachcontain a single reporter signal.

Also disclosed are methods wherein not all of the labeled proteins aredistinguished and/or separated from molecules lacking the commonproperty, not all of the reporter signals are altered, and not all ofthe altered forms of the labeled proteins are not detected at the sametime.

Also disclosed are methods wherein all of the labeled proteins aredistinguished and/or separated from molecules lacking the commonproperty, all of the reporter signals are altered, and all of thealtered forms of the labeled proteins are detected at different times.

Also disclosed are methods wherein steps (a) through (c) are performedseparately for each sample and methods wherein the different samples arefrom the same protein sample and methods wherein the different samplesare obtained at different times and methods wherein the differentsamples are from the same type of organism and methods wherein thedifferent samples are from the same type of tissue and methods whereinthe different samples are from the same organism and methods wherein thedifferent samples are obtained at different times.

Also disclosed are methods wherein the different samples are fromdifferent organisms. Also disclosed are methods wherein the differentsamples are from different types of tissues.

Also disclosed are methods wherein the different samples are fromdifferent species of organisms.

Also disclosed are methods wherein the different samples are fromdifferent strains of organisms.

Also disclosed are methods wherein the different samples are fromdifferent cellular compartments.

Also disclosed are methods further comprising identifying or preparingproteins or peptides corresponding the proteins or peptides present inone sample but not present in another sample.

Also disclosed are methods further comprising determining the relativeamount of proteins or peptides in the different samples.

Also disclosed are methods wherein the common property is one or moreaffinity tags associated with the reporter signals.

Also disclosed are methods wherein one or more affinity tags areassociated with the reporter signals.

Also disclosed are methods wherein the pattern of the presence, amount,presence and amount, or absence of labeled proteins in one of thesamples constitutes a catalog of proteins in the sample.

Also disclosed are methods wherein the pattern of the presence, amount,presence and amount, or absence of labeled proteins in a second one ofthe samples constitutes a catalog of proteins in the second sample,wherein the catalog of proteins in the first sample is a first catalogand the catalog of proteins in the second sample is a second catalog,the method further comprising comparing the first catalog and the secondcatalog.

Also disclosed are methods wherein each labeled protein comprises aprotein or a peptide and a reporter signal attached to the protein orpeptide, wherein the reporter signals comprise peptides, wherein thereporter signal peptides have the same mass-to-charge ratio.

Also disclosed are methods wherein the reporter signal peptides have thesame amino acid composition.

Also disclosed are methods wherein the reporter signal peptides have thesame amino acid sequence.

Also disclosed are methods wherein each reporter signal peptide containsa different distribution of heavy isotopes.

Also disclosed are methods wherein each reporter signal peptide containsa different distribution of substituent groups.

Also disclosed are methods wherein each reporter signal peptide has adifferent amino acid sequence.

Also disclosed are methods wherein each reporter signal peptide has alabile or scissile bond in a different location.

Also disclosed are methods wherein one or more affinity tags areassociated with the reporter signals.

Disclosed are methods comprising (a) separating a set of labeledproteins, wherein each labeled protein comprises a protein or peptideand a reporter signal attached to the protein or peptide, wherein eachlabeled protein has a common property, wherein the common propertyallows the labeled proteins comprising the same protein or peptide to bedistinguished and/or separated from molecules lacking the commonproperty, (b) altering the reporter signals, thereby altering thelabeled proteins, (c) detecting and distinguishing the altered forms ofthe labeled proteins from each other.

Disclosed are methods comprising (a) altering labeled proteins, whereineach labeled protein comprises a protein or peptide and a reportersignal attached to the protein or peptide, wherein the labeled proteinsare altered by altering the reporter signals, (b) detecting anddistinguishing the altered forms of the labeled proteins from eachother.

Disclosed are methods of detecting a protein or peptide, the methodcomprising (a) altering a labeled protein, wherein the labeled proteincomprises a protein or peptide and a reporter signal attached to theprotein or peptide, wherein the labeled protein is altered by alteringthe reporter signal, (b) detecting and distinguishing the altered formof the labeled protein from the unaltered form of labeled protein.

Also disclosed are methods further comprising, detecting the unalteredform of labeled protein.

Disclosed are methods of detecting a protein, the methods comprising,detecting a labeled protein, wherein the labeled protein comprises aprotein or peptide and a reporter signal attached to the protein orpeptide, wherein the labeled protein is altered by altering the reportersignal, detecting an altered form of the labeled protein, wherein thelabeled protein is altered by altering the reporter signal, andidentifying the protein based on the characteristics of the labeledprotein and altered form of the labeled protein.

Also disclosed are methods wherein the labeled protein and altered formof the labeled protein are detected by detecting the mass-to-chargeratio of the labeled protein and the mass-to-charge ratio of the alteredform of the labeled protein or the mass-to-charge ratio of the alteredform of the reporter signal.

Disclosed are methods comprising (a) separating one or more labeledproteins from other molecules, wherein the labeled proteins are derivedfrom one or more samples, wherein each labeled protein comprises aprotein or peptide and a reporter signal attached to the protein orpeptide, (b) altering the reporter signals, thereby altering the labeledproteins, (c) detecting and distinguishing the altered forms the labeledproteins from each other.

Also disclosed are methods further comprising, prior to step (a),associating one or more reporter signals with one or more proteins, oneor more peptides, or one or more proteins and peptides from each of theone or more samples.

Also disclosed are methods wherein steps are repeated one or more timesusing a different set of one or more reporter signals each time.

Also disclosed are methods wherein, prior to step (a), the differentsets of reporter signals are attached to proteins or peptides indifferent samples.

Also disclosed are methods wherein the different sets of reportersignals each comprise the same reporter signals.

Also disclosed are methods wherein each reporter signal or each labeledprotein has a common property, wherein the common property allows thelabeled proteins comprising the same protein or peptide to bedistinguished and/or separated from molecules lacking the commonproperty.

Also disclosed are methods wherein the one or more labeled proteins arederived from a single sample.

Also disclosed are methods wherein a single labeled protein isdistinguished and/or separated from other molecules.

Also disclosed are methods wherein a plurality of labeled proteins aredistinguished and/or separated from other molecules.

Also disclosed are methods wherein the detected altered forms of thelabeled proteins constitute a catalog of proteins in the sample.

Also disclosed are methods wherein one or more labeled proteins arederived from each of a plurality of samples.

Also disclosed are methods wherein a single labeled protein derived fromeach of the samples is distinguished and/or separated from othermolecules.

Also disclosed are methods wherein a plurality of labeled proteinsderived from each of the samples are distinguished and/or separated fromother molecules.

Also disclosed are methods wherein the detected altered forms of thelabeled proteins derived from each sample constitute a catalog ofproteins in the sample.

Disclosed are catalogs of proteins and peptides comprising, proteins andpeptides in a sample detected by (a) separating one or more labeledproteins from other molecules, wherein the labeled proteins are derivedfrom the sample, wherein each labeled protein comprises a protein orpeptide and a reporter signal attached to the protein or peptide, (b)altering the reporter signals, thereby altering the labeled proteins,(c) detecting and distinguishing the altered forms the labeled proteinsfrom each other.

Also disclosed are catalogs of proteins and peptides comprising,proteins and peptides in one or more samples detected by (a) separatingone or more labeled proteins from other molecules, wherein the labeledproteins are derived from the one or more samples, wherein each labeledprotein comprises a protein or peptide and a reporter signal attached tothe protein or peptide, (b) altering the reporter signals, therebyaltering the labeled proteins, (c) detecting and distinguishing thealtered forms the labeled proteins from each other.

C. Reporter Signal Calibration

Disclosed are methods method of producing a protein signature, themethod comprising (a) treating a protein sample to produce proteinfragments, wherein the protein fragments comprise a set of targetprotein fragments, wherein the target protein fragments can be altered,wherein the altered forms of the target protein fragments can bedistinguished from the other altered forms of the target proteinfragments, (b) mixing the target protein fragments with a set ofreporter signal calibrators, wherein each target protein fragment sharesa common property with at least one of the reporter signal calibrators,wherein the common property allows the target protein fragments andreporter signal calibrators having the common property to bedistinguished and/or separated from molecules lacking the commonproperty, wherein the target protein fragment and reporter signalcalibrator that share a common property correspond to each other,wherein the reporter signal calibrators can be altered, wherein thealtered form of each reporter signal calibrator can be distinguishedfrom the altered form of the target protein fragment with which thereporter signal calibrator shares a common property, (c) separating thetarget protein fragments and reporter signal calibrators from othermolecules based on the common properties of the target protein fragmentsand reporter signal calibrators, (d) altering the target proteinfragments and reporter signal calibrators, (e) detecting the alteredforms of the target protein fragments and reporter signal calibrators,wherein the presence, absence, amount, or presence and amount of thealtered forms of the target protein fragments indicates the presence,absence, amount, or presence and amount in the protein sample of thetarget protein fragments from which the altered forms of the targetprotein fragments are derived, wherein the presence, absence, amount, orpresence and amount of the target protein fragments in the proteinsample constitutes a protein signature of the protein sample.

Also disclosed are methods wherein steps (d) and (e) are performedsimultaneously.

Also disclosed are methods wherein the altered forms of the targetprotein fragments are detecting using mass spectrometry.

Also disclosed are methods wherein steps (c), (d), and (e) are performedwith a tandem mass spectrometer.

Also disclosed are methods wherein the tandem mass spectrometercomprises a first stage and a last stage, wherein step (c) is performedusing the first stage of the tandem mass spectrometer to select ions ina narrow mass-to-charge range, wherein step (d) is performed bycollision with a gas, and wherein step (e) is performed using the finalstage of the tandem mass spectrometer.

Also disclosed are methods where the first stage of the tandem massspectrometer is a quadrupole mass filter and methods where the finalstage of the tandem mass spectrometer is a time of flight analyzer andmethods wherein the final stage of the tandem mass spectrometer is atime of flight analyzer.

Also disclosed are methods wherein the mass-to-charge range is varied tocover the mass-to-charge ratio of each of the target protein fragments.

Also disclosed are methods wherein a predetermined amount of eachreporter signal calibrator is mixed with the target protein fragments,wherein the amount of each altered form of reporter signal calibratordetected provides a standard for assessing the amount of the alteredform of the corresponding target protein fragment.

Also disclosed are methods wherein the amount of at least two of thereporter signal calibrators is different.

Also disclosed are methods wherein the relative amount each reportersignal calibrator is based on the relative amount of each correspondingtarget protein fragment expected to be in the protein sample.

Also disclosed are methods wherein the amount of each of the reportersignal calibrators is the same.

Also disclosed are methods wherein the target protein fragments andreporter signal calibrators are altered by fragmentation.

Also disclosed are methods wherein the target protein fragments andreporter signal calibrators are altered by cleavage at a photocleavableamino acid.

Also disclosed are methods wherein the target protein fragments andreporter signal calibrators are fragmented in a collision cell.

Also disclosed are methods wherein the target protein fragments arefragmented at an asparagine-proline bond.

Also disclosed are methods wherein the protein fragments are produced byprotease digestion of the protein sample.

Also disclosed are methods wherein the protein fragments are produced bydigestion of the protein sample with a serine protease.

Also disclosed are methods wherein the serine protease is trypsin.

Also disclosed are methods wherein the protein fragments are produced bydigestion of the protein sample with Factor Xa or Enterokinase.

Also disclosed are methods wherein the protein fragments are produced bycleavage at a photocleavable amino acid.

Also disclosed are methods wherein the common property is mass-to-chargeratio, wherein the target protein fragments and reporter signalcalibrators are altered by altering their mass, their charge, or theirmass and charge, wherein the altered forms of the target proteinfragments and reporter signal calibrators can be distinguished viadifferences in the mass-to-charge ratio of the altered forms of thetarget protein fragments and reporter signal calibrators.

Also disclosed are methods wherein the set of target protein fragmentscomprises two or more, three or more, four or more, five or more, six ormore, seven or more, eight or more, nine or more, ten or more, twenty ormore, thirty or more, forty or more, fifty or more, sixty or more,seventy or more, eighty or more, ninety or more, or one hundred or moredifferent target protein fragments.

Also disclosed are methods wherein the set of target protein fragmentscomprises ten or more different target protein fragments.

Also disclosed are methods wherein the set of reporter signalcalibrators comprises two or more, three or more, four or more, five ormore, six or more, seven or more, eight or more, nine or more, ten ormore, twenty or more, thirty or more, forty or more, fifty or more,sixty or more, seventy or more, eighty or more, ninety or more, or onehundred or more different reporter signal calibrators.

Also disclosed are methods wherein the reporter signal calibratorscomprise peptides, wherein the peptides have the same mass-to-chargeratio as the corresponding target protein fragments.

Also disclosed are methods wherein the peptides have the same amino acidcomposition as the corresponding target protein fragments and methodswherein the peptides have the same amino acid sequence as thecorresponding target protein fragments and methods wherein each peptidehas a different amino acid sequence than the corresponding targetprotein fragment and methods wherein each peptide has a labile orscissile bond in a different location.

Also disclosed are methods wherein the reporter signal calibrators arepeptides, oligonucleotides, carbohydrates, polymers, oligopeptides, orpeptide nucleic acids.

Also disclosed are methods further comprising comparing the proteinsignature to one or more other protein signatures.

Also disclosed are methods wherein at least one of the target proteinfragments comprises at least one modified amino acid.

Also disclosed are methods wherein the modified amino acid is aphosphorylated amino acid, an acylated amino acid, or a glycosylatedamino acid.

Also disclosed are methods wherein at least one of the target proteinfragments is the same as the target protein fragment comprising themodified amino acid except for the modified amino acid.

Also disclosed are methods further comprising performing steps (a)through (e) on a plurality of protein samples.

Also disclosed are methods further comprising identifying differencesbetween the protein signatures produced from the protein samples.

Also disclosed are methods further comprising performing steps (a)through (e) on a control protein sample, identifying differences betweenthe protein signatures produced from the protein samples and the controlprotein sample.

Also disclosed are methods wherein the differences are differences inthe presence, amount, presence and amount, or absence of target proteinfragments in the protein samples and the control protein sample.

Also disclosed are methods wherein the steps (a) through (e) areperformed on a control protein sample and a tester protein sample,wherein the tester protein sample, or the source of the tester proteinsample, is treated, prior to step (a), so as to destroy, disrupt oreliminate one or more protein molecules in the tester protein sample,wherein the target protein fragments corresponding to the destroyed,disrupted, or eliminated protein molecules will be produced from thecontrol protein sample but not the tester protein sample.

Also disclosed are methods wherein the tester protein sample is treatedso as to destroy, disrupt or eliminate one or more protein molecules inthe tester protein sample.

Also disclosed are methods wherein one or more protein molecules in thetester sample are eliminated by separating the one or more proteinmolecules from the tester protein sample.

Also disclosed are methods wherein the one or more protein molecules areseparated by affinity separation.

Also disclosed are methods wherein the source of the tester proteinsample is treated so as to destroy, disrupt or eliminate one or moreprotein molecules in the tester protein sample.

Also disclosed are methods wherein the treatment of the source isaccomplished by exposing cells from which the tester sample will bederived with a compound, composition, or condition that will reduce oreliminate expression of one or more genes.

Also disclosed are methods further comprising identifying differences inthe target protein fragments in the control protein sample and testerprotein sample.

Also disclosed are methods further comprising identifying differencesbetween the target protein fragments in the protein samples.

Also disclosed are methods wherein the plurality of protein samples areproduced by a separation procedure, wherein the separation procedurecomprises liquid chromatography, gel electrophoresis, two-dimensionalchromatography, two-dimensional gel electrophoresis, isoelectricfocusing, thin layer chromatography, centrifugation, filtration, ionchromatography, immunoaffinity chromatography, membrane separation, or acombination of these.

Also disclosed are methods wherein the protein samples are differentfractions or samples produced by the same separation procedure.

Also disclosed are methods further comprising performing steps (a)through (e) on a second protein sample.

Also disclosed are methods wherein the second protein sample is a samplefrom the same type of organism as the first protein sample.

Also disclosed are methods wherein the second protein sample is a samplefrom the same type of tissue as the first protein sample.

Also disclosed are methods wherein the second protein sample is a samplefrom the same organism as the first protein sample.

Also disclosed are methods wherein the second protein sample is obtainedat a different time than the first protein sample.

Also disclosed are methods wherein the second protein sample is a samplefrom a different organism than the first protein sample.

Also disclosed are methods wherein the second protein sample is a samplefrom a different type of tissue than the first protein sample.

Also disclosed are methods wherein the second protein sample is a samplefrom a different species of organism than the first protein sample.

Also disclosed are methods wherein the second protein sample is a samplefrom a different strain of organism than the first protein sample.

Also disclosed are methods wherein the second protein sample is a samplefrom a different cellular compartment than the first protein sample.

Also disclosed are methods further comprising producing a second proteinsignature from a second protein sample and comparing the first proteinsignature and second protein signature, wherein differences in the firstand second protein signatures indicate differences in source orcondition of the source of the first and second protein samples.

Also disclosed are methods further comprising producing a second proteinsignature from a second protein sample and comparing the first proteinsignature and second protein signature, wherein differences in the firstand second protein signatures indicate differences in proteinmodification of the first and second protein samples.

Also disclosed are methods wherein the second protein sample is a samplefrom the same type of cells as the first protein sample except that thecells from which the first protein sample is derived aremodification-deficient relative to the cells from which the secondprotein sample is derived.

Also disclosed are methods wherein the second protein sample is a samplefrom a different type of cells than the first protein sample, andwherein the cells from which the first protein sample is derived aremodification-deficient relative to the cells from which the secondprotein sample is derived.

Also disclosed are methods wherein the protein sample is derived fromone or more cells.

Also disclosed are methods wherein the protein signature indicates thephysiological state of the cells.

Also disclosed are methods wherein the protein signature indicates theeffect of a treatment of the cells.

Also disclosed are methods wherein the cells are derived from anorganism, wherein the cells are treated by treating the organism.

Also disclosed are methods wherein the organism is treated byadministering a compound to the organism.

Also disclosed are methods wherein the organism is human.

Also disclosed are methods wherein the protein sample is produced by aseparation procedure, wherein the separation procedure comprises liquidchromatography, gel electrophoresis, two-dimensional chromatography,two-dimensional gel electrophoresis, isoelectric focusing, thin layerchromatography, centrifugation, filtration, ion chromatography,immunoaffinity chromatography, membrane separation, or a combination ofthese.

Also disclosed are methods wherein the set of reporter signalcalibrators consists of a single reporter signal calibrator.

Also disclosed are methods wherein the protein signature of the proteinsample represents the presence, absence, amount, or presence and amountof the target protein fragment in the protein sample that corresponds tothe reporter signal calibrator.

Disclosed are methods of producing a protein signature, the methodcomprising detecting altered forms of target protein fragments andreporter signal calibrators, wherein the altered forms of the targetprotein fragments can be distinguished from the other altered forms ofthe target protein fragments, wherein each target protein fragmentshares a common property with at least one of the reporter signalcalibrators, wherein the common property allows the target proteinfragments and reporter signal calibrators having the common property tobe distinguished and/or separated from molecules lacking the commonproperty, wherein the target protein fragment and reporter signalcalibrator that share a common property correspond to each other,wherein the altered form of each reporter signal calibrator can bedistinguished from the altered form of the target protein fragment withwhich the reporter signal calibrator shares a common property, whereinthe presence, absence, amount, or presence and amount of the alteredforms of the target protein fragments indicates the presence, absence,amount, or presence and amount in a protein sample of the target proteinfragments from which the altered forms of the target protein fragmentsare derived, wherein the presence, absence, amount, or presence andamount of the target protein fragments in the protein sample constitutesa protein signature of the protein sample.

Also disclosed are methods wherein the target protein fragments andreporter signal calibrators are distinguished and/or separated fromother molecules based on the common properties of the target proteinfragments and reporter signal calibrators.

Also disclosed are methods wherein the target protein fragments andreporter signal calibrators are altered following separation.

Also disclosed are methods wherein the target protein fragments areproduced by treating the protein sample.

Disclosed are methods of producing a protein signature, the methodcomprising (a) treating a protein sample to produce protein fragments,wherein the protein fragments comprise a set of target proteinfragments, wherein the target protein fragments can be altered, whereinthe altered forms of the target protein fragments can be distinguishedfrom the other altered forms of the target protein fragments, (b)separating the target protein fragments from other protein fragments inthe protein sample, (c) altering the target protein fragments, (d)detecting the altered forms of the target protein fragments, wherein thepresence, absence, amount, or presence and amount of the altered formsof the target protein fragments indicates the presence, absence, amount,or presence and amount in the protein sample of the target proteinfragments from which the altered forms of the target protein fragmentsare derived, wherein the presence, absence, amount, or presence andamount of the target protein fragments in the protein sample constitutesa protein signature of the protein sample.

Also disclosed are methods further comprising, prior to or simultaneouswith step (b), mixing the target protein fragments with a set ofreporter signal calibrators, wherein each target protein fragment sharesa common property with at least one of the reporter signal calibrators,wherein the common property allows the target protein fragments andreporter signal calibrators having the common property to bedistinguished and/or separated from molecules lacking the commonproperty, wherein the reporter signal calibrators can be altered,wherein the altered form of each reporter signal calibrator can bedistinguished from the altered form of the target protein fragment withwhich the reporter signal calibrator shares a common property.

Disclosed is a method of producing a protein signature, the methodcomprising (a) separating a plurality of target protein fragments fromother protein fragments in a protein sample, (b) altering the targetprotein fragments, (c) detecting the altered forms of the target proteinfragments, wherein the presence, absence, amount, or presence and amountof the altered forms of the target protein fragments indicates thepresence, absence, amount, or presence and amount in the protein sampleof the target protein fragments from which the altered forms of thetarget protein fragments are derived, wherein the presence, absence,amount, or presence and amount of the target protein fragments in theprotein sample constitutes a protein signature of the protein sample.

Disclosed are methods of analyzing a protein sample, the methodcomprising (a) mixing a protein sample with a predetermined amount of areporter signal calibrator, wherein the protein sample has a knownamount of protein, wherein the protein sample comprises a target proteinfragment, wherein the target protein fragment can be altered, whereinthe reporter signal calibrator can be altered, wherein the altered formof the reporter signal calibrator can be distinguished from the alteredform of the target protein fragment, (b) altering the target proteinfragment and reporter signal calibrator, (c) detecting the altered formsof the target protein fragment and reporter signal calibrator.

Also disclosed are methods further comprising determining the ratio ofthe amount of the target protein fragment and the amount of the reportersignal calibrator detected, and comparing the determined ratio with thepredicted ratio of the amount of the target protein fragment and theamount of the reporter signal calibrator, wherein the predicted ratio isbased on the predicted amount of target protein fragment in the proteinsample and the predetermined amount of reporter signal calibrator,wherein the predicted amount of target protein fragment is the amount oftarget protein fragment the protein sample would have if the knownamount of protein in the protein sample consisted of the target proteinfragment, wherein the difference between the determined ratio and thepredicted ratio is a measure of the purity of the protein sample for thetarget protein fragment, wherein the closer the determined ratio is tothe predicted ratio, the purer the protein sample.

Disclosed are methods of analyzing a protein sample, the methodcomprising (a) treating a protein sample to produce protein fragments,wherein the protein sample has a known amount of protein, wherein theprotein sample comprises a target protein, wherein the protein fragmentscomprise a target protein fragment derived from the target protein, (b)mixing the protein sample with a predetermined amount of a reportersignal calibrator, wherein the target protein fragment can be altered,wherein the reporter signal calibrator can be altered, wherein thealtered form of the reporter signal calibrator can be distinguished fromthe altered form of the target protein fragment, (b) altering the targetprotein fragment and reporter signal calibrator, (c) detecting thealtered forms of the target protein fragment and reporter signalcalibrator.

Also disclosed are methods further comprising determining the ratio ofthe amount of the target protein fragment and the amount of the reportersignal calibrator detected, and comparing the determined ratio with thepredicted ratio of the amount of the target protein fragment and theamount of the reporter signal calibrator, wherein the predicted ratio isbased on the predicted amount of target protein fragment in the proteinsample and the predetermined amount of reporter signal calibrator,wherein the predicted amount of target protein fragment is the amount oftarget protein fragment the protein sample would have if the knownamount of protein in the protein sample consisted of the target protein,wherein the difference between the determined ratio and the predictedratio is a measure of the purity of the protein sample for the targetprotein, wherein the closer the determined ratio is to the predictedratio, the purer the protein sample.

Disclosed are methods of producing a protein signature, the methodcomprising (a) treating a protein sample to produce protein fragments,wherein the protein fragments comprise a set of target proteinfragments, wherein each of the target protein fragments can be altered,wherein the altered forms of each target protein fragment can bedistinguished from every other altered form of target protein fragment,(b) mixing the target protein fragments with a set of reporter signalcalibrators, wherein each target protein fragment shares a commonproperty with at least one of the reporter signal calibrators, whereinthe common property allows each of the target protein fragments andreporter signal calibrators having the common property to bedistinguished and/or separated from molecules lacking the commonproperty, wherein the target protein fragment and reporter signalcalibrator that share a common property correspond to each other,wherein each of the reporter signal calibrators can be altered, whereinthe altered form of each reporter signal calibrator can be distinguishedfrom the altered form of the target protein fragment with which thereporter signal calibrator shares a common property, (c) separating thetarget protein fragments and reporter signal calibrators from othermolecules based on the common properties of the target protein fragmentsand reporter signal calibrators, (d) altering the target proteinfragments and reporter signal calibrators, (e) detecting the alteredforms of the target protein fragments and reporter signal calibrators,wherein the presence, absence, amount, or presence and amount of thealtered forms of the target protein fragments indicates the presence,absence, amount, or presence and amount in the protein sample of thetarget protein fragments from which the altered forms of the targetprotein fragments are derived, wherein the presence, absence, amount, orpresence and amount of the target protein fragments in the proteinsample constitutes a protein signature of the protein sample.

Disclosed are methods of producing a protein signature, the methodcomprising detecting altered forms of target protein fragments andreporter signal calibrators, wherein the altered forms of each targetprotein fragment can be distinguished from every other altered form oftarget protein fragment, wherein each target protein fragment shares acommon property with at least one of the reporter signal calibrators,wherein the common property allows each of the target protein fragmentsand reporter signal calibrators having the common property to bedistinguished and/or separated from molecules lacking the commonproperty, wherein the target protein fragment and reporter signalcalibrator that share a common property correspond to each other,wherein the altered form of each reporter signal calibrator can bedistinguished from the altered form of the target protein fragment withwhich the reporter signal calibrator shares a common property, whereinthe presence, absence, amount, or presence and amount of the alteredforms of the target protein fragments indicates the presence, absence,amount, or presence and amount in a protein sample of the target proteinfragments from which the altered forms of the target protein fragmentsare derived, wherein the presence, absence, amount, or presence andamount of the target protein fragments in the protein sample constitutesa protein signature of the protein sample.

Disclosed are methods of producing a protein signature, the methodcomprising (a) treating a protein sample to produce protein fragments,wherein the protein fragments comprise a set of target proteinfragments, wherein each of the target protein fragments can be altered,wherein the altered forms of each target protein fragment can bedistinguished from every other altered form of target protein fragment,(b) separating the target protein fragments from other protein fragmentsin the protein sample, (c) altering the target protein fragments, (d)detecting the altered forms of the target protein fragments, wherein thepresence, absence, amount, or presence and amount of the altered formsof the target protein fragments indicates the presence, absence, amount,or presence and amount in the protein sample of the target proteinfragments from which the altered forms of the target protein fragmentsare derived, wherein the presence, absence, amount, or presence andamount of the target protein fragments in the protein sample constitutesa protein signature of the protein sample.

Also disclosed are methods further comprising, prior to or simultaneouswith step (b), mixing the target protein fragments with a set ofreporter signal calibrators, wherein each target protein fragment sharesa common property with at least one of the reporter signal calibrators,wherein the common property allows each of the target protein fragmentsand reporter signal calibrators having the common property to bedistinguished and/or separated from molecules lacking the commonproperty, wherein each of the reporter signal calibrators can bealtered, wherein the altered form of each reporter signal calibrator canbe distinguished from the altered form of the target protein fragmentwith which the reporter signal calibrator shares a common property.

D. Reporter Signal Fusions

Disclosed are methods of detecting expression, the method comprisingdetecting a target altered reporter signal peptide derived from one ormore expression samples, wherein the one or more expression samplescollectively comprise a set of nucleic acid molecules, wherein eachnucleic acid molecule comprises a nucleotide segment encoding an aminoacid segment comprising a reporter signal peptide and a protein orpeptide of interest, wherein the reporter signal peptides have a commonproperty, wherein the common property allows the reporter signalpeptides to be distinguished and/or separated from molecules lacking thecommon property, wherein the reporter signal peptides can be altered,wherein the altered form of each reporter signal peptide can bedistinguished from the altered forms of the other reporter signalpeptides, wherein the target altered reporter signal peptide is one ofthe altered reporter signal peptides, wherein detection of the targetaltered reporter signal peptide indicates expression of the amino acidsegment that comprises the reporter signal peptide from which the targetaltered reporter signal peptide is derived.

Also disclosed are methods further comprising determining the amount ofthe target altered reporter signal peptide detected, wherein the amountof the target altered reporter signal peptide indicates the amountpresent in the one or more expression samples of the amino acid segmentthat comprises the reporter signal peptide from which the target alteredreporter signal peptide is derived.

Also disclosed are methods wherein the amount of the amino acid segmentpresent is proportional to the amount of the target altered reportersignal peptide detected.

Also disclosed are methods further comprising detecting a plurality ofthe altered reporter signal peptides, wherein detection of each alteredreporter signal peptide indicates expression of the amino acid segmentthat comprises the reporter signal peptide from which that alteredreporter signal peptide is derived.

Also disclosed are methods further comprising determining the amount ofthe altered reporter signal peptides detected, wherein the amount ofeach altered reporter signal peptide indicates the amount present in theone or more expression samples of the amino acid segment that comprisesthe reporter signal peptide from which that altered reporter signalpeptide is derived.

Also disclosed are methods wherein the amount of the amino acid segmentpresent is proportional to the amount of the altered reporter signalpeptide detected.

Also disclosed are methods wherein the presence, absence, amount, orpresence and amount of the altered forms of the reporter signal peptidesindicates the presence, absence, amount, or presence and amount in theexpression sample of the reporter signal peptides from which the alteredforms of the reporter signal peptides are derived, wherein the presence,absence, amount, or presence and amount of the reporter signal peptidesin the expression sample constitutes a protein signature of theexpression sample.

Also disclosed are methods wherein the altered forms of the reportersignal peptides are detecting using mass spectrometry.

Also disclosed are methods wherein the altered forms of the reportersignal peptides are detected with a tandem mass spectrometer.

Also disclosed are methods wherein the mass spectrometer includes aquadrupole set for single-ion filtering, a collision cell, and atime-of-flight spectrometer.

Also disclosed are methods wherein the reporter signal peptides arealtered by fragmentation.

Also disclosed are methods wherein the reporter signal peptides arealtered by cleavage at a photocleavable amino acid.

Also disclosed are methods wherein the reporter signal peptides arefragmented in a collision cell.

Also disclosed are methods wherein the reporter signal peptides arefragmented at an asparagine-proline bond, a methionine, or aphosphorylated amino acid.

Also disclosed are methods wherein the common property is mass-to-chargeratio, wherein the reporter signal peptides are altered by alteringtheir mass, their charge, or their mass and charge, wherein the alteredforms of the reporter signal peptides can be distinguished viadifferences in the mass-to-charge ratio of the altered forms of thereporter signal peptides.

Also disclosed are methods wherein there are two or more, three or more,four or more, five or more, six or more, seven or more, eight or more,nine or more, ten or more, twenty or more, thirty or more, forty ormore, fifty or more, sixty or more, seventy or more, eighty or more,ninety or more, or one hundred or more different reporter signalpeptides.

Also disclosed are methods wherein there are ten or more differentreporter signal peptides.

Also disclosed are methods wherein each peptide has a labile or scissilebond in a different location.

Also disclosed are methods further comprising comparing the proteinsignature to one or more other protein signatures.

Also disclosed are methods wherein the detected altered reporter signalpeptides are derived from a plurality of expression samples.

Also disclosed are methods wherein some of the detected altered reportersignal peptides derived from a control expression sample, identifyingdifferences between the protein signatures produced from the expressionsamples and the control expression sample.

Also disclosed are methods wherein the differences are differences inthe presence, amount, presence and amount, or absence of reporter signalpeptides in the expression samples and the control expression sample.

Also disclosed are methods wherein the plurality of expression samplescomprises a control expression sample and a tester expression sample,wherein the tester expression sample, or the source of the testerexpression sample, is treated so as to destroy, disrupt or eliminate oneor more of the amino acid segments in the tester expression sample,wherein the reporter signal peptides corresponding to the destroyed,disrupted, or eliminated amino acid segments will be produced from thecontrol expression sample but not the tester expression sample.

Also disclosed are methods wherein the tester expression sample istreated so as to destroy, disrupt or eliminate one or more of the aminoacid segments in the tester expression sample.

Also disclosed are methods wherein one or more of the amino acidsegments in the tester sample are eliminated by separating the one ormore of the amino acid segments from the tester expression sample.

Also disclosed are methods wherein the one or more of the amino acidsegments are separated by affinity separation.

Also disclosed are methods wherein the source of the tester expressionsample is treated so as to destroy, disrupt or eliminate one or more ofthe amino acid segments in the tester expression sample.

Also disclosed are methods wherein the treatment of the source isaccomplished by exposing cells from which the tester sample will bederived with a compound, composition, or condition that will reduce oreliminate expression of one or more of the nucleotide segments.

Also disclosed are methods further comprising identifying differences inthe reporter signal peptides in the control expression sample and testerexpression sample.

Also disclosed are methods further comprising identifying differencesbetween the reporter signal peptides in the expression samples.

Also disclosed are methods wherein at least two of the expressionsamples, or the sources of the at least two expression samples, aresubjected to different conditions.

Also disclosed are methods wherein the sources of the expression samplesare cells.

Also disclosed are methods wherein differences in the protein signaturesof the at least two expression samples indicate the effect of thedifferent conditions.

Also disclosed are methods wherein the different conditions are exposureto different compounds.

Also disclosed are methods wherein the different conditions are exposureto a compound and no exposure to the compound.

Also disclosed are methods further comprising producing a second proteinsignature from a second expression sample and comparing the firstprotein signature and second protein signature, wherein differences inthe first and second protein signatures indicate differences in sourceor condition of the source of the first and second expression samples.

Also disclosed are methods further comprising producing a second proteinsignature from a second expression sample and comparing the firstprotein signature and second protein signature, wherein differences inthe first and second protein signatures indicate differences in proteinmodification of the first and second expression samples.

Also disclosed are methods wherein the second expression sample is asample from the same type of cells as the first expression sample exceptthat the cells from which the first expression sample is derived aremodification-deficient relative to the cells from which the secondexpression sample is derived.

Also disclosed are methods wherein the second expression sample is asample from a different type of cells than the first expression sample,and wherein the cells from which the first expression sample is derivedare modification-deficient relative to the cells from which the secondexpression sample is derived.

Also disclosed are methods wherein the expression sample is derived fromone or more cells.

Also disclosed are methods wherein the protein signature indicates thephysiological state of the cells.

Also disclosed are methods wherein the protein signature indicates theeffect of a treatment of the cells.

Also disclosed are methods wherein the cells are derived from anorganism, wherein the cells are treated by treating the organism.

Also disclosed are methods wherein the organism is treated byadministering a compound to the organism.

Also disclosed are methods wherein the organism is human.

Also disclosed are methods wherein altered reporter signal peptides aredetected in a first and a second expression sample.

Also disclosed are methods wherein the second expression sample is asample from the same type of organism as the first expression sample.

Also disclosed are methods wherein the second expression sample is asample from the same type of tissue as the first expression sample.

Also disclosed are methods wherein the second expression sample is asample from the same organism as the first expression sample.

Also disclosed are methods wherein the second expression sample isobtained at a different time than the first expression sample.

Also disclosed are methods wherein the second expression sample is asample from a different organism than the first expression sample.

Also disclosed are methods wherein the second expression sample is asample from a different type of tissue than the first expression sample.

Also disclosed are methods wherein the second expression sample is asample from a different species of organism than the first expressionsample.

Also disclosed are methods wherein the second expression sample is asample from a different strain of organism than the first expressionsample.

Also disclosed are methods wherein the second expression sample is asample from a different cellular compartment than the first expressionsample.

Also disclosed are methods further comprising altering the reportersignal peptides.

Also disclosed are methods wherein the reporter signal peptides arealtered by fragmentation.

Also disclosed are methods wherein the reporter signal peptides arealtered by cleavage at a photocleavable amino acid.

Also disclosed are methods wherein the reporter signal peptides arefragmented in a collision cell.

Also disclosed are methods wherein the reporter signal peptides arefragmented at an asparagine-proline bond, a methionine, or aphosphorylated amino acid.

Also disclosed are methods further comprising separating the reportersignal peptides from the expression samples.

Also disclosed are methods wherein the reporter signal peptides aredistinguished and/or separated from the expression samples based on thecommon property.

Also disclosed are methods further comprising cleaving the reportersignal peptides from the proteins or peptides of interest.

Also disclosed are methods wherein the reporter signal peptides aredistinguished and/or separated from the proteins or peptides of interestbased on the common property.

Also disclosed are methods further comprising cleaving the amino acidsegments into a reporter signal peptide portion and a protein portion.

Also disclosed are methods further comprising mixing two or more of theexpression samples together.

Also disclosed are methods further comprising mixing two or more aminoacid segments together, wherein the mixed amino acid segments werederived from two or more different expression samples.

Also disclosed are methods wherein expression of the amino acid segmentthat comprises the reporter signal peptide from which the target alteredreporter signal peptide is derived identifies the expression sample fromwhich the target altered reporter signal peptide is derived.

Also disclosed are methods wherein the expression samples are derivedfrom one or more cells, wherein expression of the amino acid segmentthat comprises the reporter signal peptide from which the target alteredreporter signal peptide is derived identifies the cell from which theidentified expression sample is derived.

Also disclosed are methods wherein the expression samples are derivedfrom one or more organisms, wherein expression of the amino acid segmentthat comprises the reporter signal peptide from which the target alteredreporter signal peptide is derived identifies the organism from whichthe identified expression sample is derived.

Also disclosed are methods wherein the expression samples are derivedfrom one or more tissues, wherein expression of the amino acid segmentthat comprises the reporter signal peptide from which the target alteredreporter signal peptide is derived identifies the tissue from which theidentified expression sample is derived.

Also disclosed are methods wherein the expression samples are derivedfrom one or more cell lines, wherein expression of the amino acidsegment that comprises the reporter signal peptide from which the targetaltered reporter signal peptide is derived identifies the cell line fromwhich the identified expression sample is derived.

Also disclosed are methods wherein each nucleic acid molecule furthercomprises expression sequences, wherein the expression sequences areoperably linked to the nucleotide segment such that the amino acidsegment is expressed.

Also disclosed are methods wherein the expression sequences comprisetranslation expression sequences.

Also disclosed are methods wherein the expression sequences furthercomprise transcription expression sequences.

Also disclosed are methods wherein the amino acid segment is expressedin vitro.

Also disclosed are methods wherein the amino acid segment is expressedin vivo.

Also disclosed are methods wherein the amino acid segment is expressedin cell culture.

Also disclosed are methods wherein the expression sequences of eachnucleic acid molecule are different.

Also disclosed are methods wherein the different expression sequencesare differently regulated.

Also disclosed are methods wherein the expression sequences aresimilarly regulated.

Also disclosed are methods wherein a plurality of the expressionsequences are expression sequences of, or derived from, genes expressedas part of the same expression cascade.

Also disclosed are methods wherein the expression sequences of eachnucleic acid molecule are the same.

Also disclosed are methods wherein the expression sequences aresimilarly regulated.

Also disclosed are methods wherein the expression sequences of at leasttwo nucleic acid molecules are different.

Also disclosed are methods wherein the expression sequences of at leasttwo nucleic acid molecules are the same.

Also disclosed are methods wherein expression of the amino acid segmentis induced.

Also disclosed are methods wherein each nucleic acid molecule furthercomprises replication sequences, wherein the replication sequencesmediate replication of the nucleic acid molecules.

Also disclosed are methods wherein the nucleic acid molecules arereplicated in vitro.

Also disclosed are methods wherein the nucleic acid molecules arereplicated in vivo.

Also disclosed are methods wherein the nucleic acid molecules arereplicated in cell culture.

Also disclosed are methods wherein each nucleic acid molecule furthercomprises integration sequences, wherein the integration sequencesmediate integration of the nucleic acid molecules into other nucleicacids.

Also disclosed are methods wherein the nucleic acid molecules areintegrated into a chromosome.

Also disclosed are methods wherein the nucleic acid molecules areintegrated into a chromosome at a predetermined location.

Also disclosed are methods wherein the nucleic acids molecules areproduced by replicating nucleic acids in one or more nucleic acidsamples.

Also disclosed are methods wherein the nucleic acids are replicatedusing pairs of primers, wherein each of the first primers in the primerpairs used to produce the nucleic acid molecules comprises a nucleotidesequence encoding the reporter signal peptide.

Also disclosed are methods wherein each first primer further comprisesexpression sequences.

Also disclosed are methods wherein the nucleotide sequence of each firstprimer also encodes an epitope tag.

Also disclosed are methods wherein each amino acid segment furthercomprises an epitope tag.

Also disclosed are methods wherein the epitope tag of each amino acidsegment is different.

Also disclosed are methods wherein the epitope tag of each amino acidsegment is the same.

Also disclosed are methods wherein the epitope tag of at least two aminoacid segments are different.

Also disclosed are methods wherein the epitope tag of at least two aminoacid segments are the same.

Also disclosed are methods wherein the amino acid segments aredistinguished and/or separated from the one or more expression samplesvia the epitope tags.

Also disclosed are methods wherein the reporter signal peptide of eachamino acid segment is different.

Also disclosed are methods wherein the reporter signal peptide of eachamino acid segment is the same.

Also disclosed are methods wherein the reporter signal peptide of atleast two amino acid segments are different.

Also disclosed are methods wherein the reporter signal peptide of atleast two amino acid segments are the same.

Also disclosed are methods wherein the nucleic acid molecules are incells.

Also disclosed are methods wherein each nucleic acid molecule is in adifferent cell.

Also disclosed are methods wherein each nucleic acid molecule is in thesame cell.

Also disclosed are methods wherein each nucleic acid molecule furthercomprises expression sequences, wherein the expression sequences areoperably linked to the nucleotide segment such that the amino acidsegment can be expressed.

Also disclosed are methods wherein the expression sequences of eachnucleic acid molecule are different.

Also disclosed are methods wherein the expression sequences aresimilarly regulated.

Also disclosed are methods wherein a plurality of the expressionsequences are expression sequences of, or derived from, genes expressedas part of the same expression cascade.

Also disclosed are methods wherein the nucleic acid molecules areintegrated into a chromosome of the cell.

Also disclosed are methods wherein the nucleic acid molecules areintegrated into the chromosome at a predetermined location.

Also disclosed are methods wherein the chromosome is an artificialchromosome.

Also disclosed are methods wherein the nucleic acid molecules are, orare integrated into, a plasmid.

Also disclosed are methods wherein the cells are in cell lines.

Also disclosed are methods wherein each nucleic acid molecule is in adifferent cell line.

408H. The method of claim 408F wherein each nucleic acid molecule is inthe same cell line.

Also disclosed are methods wherein the expression samples are producedfrom the cells.

Also disclosed are methods wherein each expression sample is producedfrom cells from a cell sample, wherein each expression sample isproduced from a different cell sample.

Also disclosed are methods wherein each cell sample is subjected todifferent conditions.

Also disclosed are methods wherein each cell sample is brought intocontact with a different test compound.

Also disclosed are methods wherein each cell sample is cultured underdifferent conditions.

Also disclosed are methods wherein each cell sample is derived from adifferent organism.

Also disclosed are methods wherein each cell sample is derived from adifferent tissue.

Also disclosed are methods wherein each cell sample is taken from thesame source at different times.

Also disclosed are methods wherein the expression samples are producedby lysing the cells.

Also disclosed are methods wherein the nucleic acid molecules are inorganisms.

Also disclosed are methods wherein each nucleic acid molecule is in adifferent organism.

Also disclosed are methods wherein each nucleic acid molecule is in thesame organism.

Also disclosed are methods wherein each nucleic acid molecule furthercomprises expression sequences, wherein the expression sequences areoperably linked to the nucleotide segment such that the amino acidsegment can be expressed.

Also disclosed are methods wherein the expression sequences of eachnucleic acid molecule are different.

Also disclosed are methods wherein the expression sequences aresimilarly regulated.

Also disclosed are methods wherein a plurality of the expressionsequences are expression sequences of, or derived from, genes expressedas part of the same expression cascade.

Also disclosed are methods wherein the nucleic acid molecules areintegrated into a chromosome of the organism.

Also disclosed are methods wherein the nucleic acid molecules areintegrated into the chromosome at a predetermined location.

Also disclosed are methods wherein the chromosome is an artificialchromosome.

Also disclosed are methods wherein the nucleic acid molecules are, orare integrated into, a plasmid.

Also disclosed are methods wherein each nucleic acid molecule is in adifferent organism.

Also disclosed are methods wherein each nucleic acid molecule is in thesame organism.

Also disclosed are methods wherein the nucleic acid molecules are incells of an organism.

Also disclosed are methods wherein the nucleic acid molecules are insubstantially all of the cells of the organism.

Also disclosed are methods wherein the nucleic acid molecules are insome of the cells of the organism.

Also disclosed are methods wherein the amino acid segments are expressedin substantially all of the cells of the organism.

Also disclosed are methods wherein the amino acid segments are expressedin some of the cells of the organism.

Also disclosed are methods wherein the protein or peptide of interest ofeach amino acid segment is different.

Also disclosed are methods wherein the protein or peptide of interest ofeach amino acid segment is the same.

Also disclosed are methods wherein the protein or peptide of interest ofat least two amino acid segments are different.

Also disclosed are methods wherein the protein or peptide of interest ofat least two amino acid segments are the same.

Also disclosed are methods wherein the proteins or peptides of interestare related.

Also disclosed are methods wherein the proteins or peptides of interestare proteins produced in the same cascade.

Also disclosed are methods wherein the proteins or peptides of interestare proteins expressed under the same conditions.

Also disclosed are methods wherein the proteins or peptides of interestare proteins associated with the same disease.

Also disclosed are methods wherein the proteins or peptides of interestare proteins associated with the same cell type.

Also disclosed are methods wherein the proteins or peptides of interestare proteins associated with the same tissue type.

Also disclosed are methods wherein the proteins or peptides of interestare proteins in the same enzymatic pathway.

Also disclosed are methods wherein the nucleotide segment encodes aplurality of amino acid segments each comprising a reporter signalpeptide and a protein or peptide of interest.

Also disclosed are methods wherein the protein or peptide of interest ofat least two of the amino acid segments in one of the nucleotidesegments are different.

Also disclosed are methods wherein the protein or peptide of interest ofthe amino acid segments in one of the nucleotide segments are different.

Also disclosed are methods wherein the protein or peptide of interest ofat least two of the amino acid segments in each of the nucleotidesegments are different.

Also disclosed are methods wherein the protein or peptide of interest ofthe amino acid segments in each of the nucleotide segments aredifferent.

Also disclosed are methods wherein the set consists of a single nucleicacid molecule.

Also disclosed are methods wherein the set consists of a single nucleicacid molecule, wherein the nucleic acid molecule comprises a pluralityof nucleotide segments each encoding an amino acid segment.

Also disclosed are methods wherein the amino acid segment comprises acleavage site near the junction between the reporter signal peptide andthe protein or peptide of interest.

Also disclosed are methods wherein the cleavage site is cleaved.

Also disclosed are methods wherein the reporter signal peptide isdistinguished and/or separated from the peptide or protein of interest.

Also disclosed are methods wherein the cleavage site is a trypsincleavage site.

Also disclosed are methods wherein the cleavage site is at the junctionbetween the reporter signal peptide and the protein or peptide ofinterest.

Also disclosed are methods wherein each amino acid segment furthercomprises a self-cleaving segment.

Also disclosed are methods wherein the self-cleaving segment is betweenthe reporter signal peptide and the protein or peptide of interest.

Also disclosed are methods wherein the self-cleaving segment cleaves theamino acid segment.

Also disclosed are methods wherein the reporter signal peptide isdistinguished and/or separated from the peptide or protein of interest.

Also disclosed are methods wherein the self-cleaving segment is anintein segment.

Also disclosed are methods wherein a plurality of different alteredreporter signal peptides are detected, wherein detection of each alteredreporter signal peptide indicates expression of the amino acid segmentthat comprises the reporter signal peptide from which that alteredreporter signal peptide is derived.

Also disclosed are methods wherein different expression samples comprisedifferent nucleic acid molecules, wherein detection of each alteredreporter signal peptide indicates expression in the expression samplethat comprises the nucleic acid molecule that comprises the nucleotidesegment encoding the amino acid segment that comprises the reportersignal peptide from which that altered reporter signal peptide isderived.

Also disclosed are methods wherein there are a plurality of differentexpression samples, wherein each different expression sample comprisesdifferent nucleic acid molecules, wherein detection of an alteredreporter signal peptide indicates expression in the expression samplethat comprises the nucleic acid molecule that comprises the nucleotidesegment encoding the amino acid segment that comprises the reportersignal peptide from which the detected altered reporter signal peptideis derived.

Disclosed are methods of detecting expression, the method comprisingdetecting a target altered reporter signal peptide derived from one ormore expression samples, wherein the one or more expression samplescollectively comprise a set of nucleic acid molecules, wherein eachnucleic acid molecule comprises a nucleotide segment encoding an aminoacid segment comprising a reporter signal peptide and a protein orpeptide of interest, wherein the reporter signal peptides have a commonproperty, wherein the common property allows the reporter signalpeptides to be distinguished and/or separated from molecules lacking thecommon property, wherein the reporter signal peptides can be altered,wherein the altered form of each reporter signal peptide can bedistinguished from the altered forms of the other reporter signalpeptides, wherein the target altered reporter signal peptide is one ofthe altered reporter signal peptides, wherein detection of the targetaltered reporter signal peptide indicates expression of the nucleotidesegment encoding the amino acid segment that comprises the reportersignal peptide from which the target altered reporter signal peptide isderived.

Also disclosed are methods further comprising determining the amount ofthe target altered reporter signal peptide detected, wherein the amountof the target altered reporter signal peptide indicates the amountpresent in the one or more expression samples of the nucleotide segmentthat comprises the reporter signal peptide from which the target alteredreporter signal peptide is derived.

Also disclosed are methods wherein the amount of the nucleotide segmentpresent is proportional to the amount of the target altered reportersignal peptide detected.

Also disclosed are methods further comprising detecting a plurality ofthe altered reporter signal peptides, wherein detection of each alteredreporter signal peptide indicates expression of the nucleotide segmentthat comprises the reporter signal peptide from which that alteredreporter signal peptide is derived.

Also disclosed are methods further comprising determining the amount ofthe altered reporter signal peptides detected, wherein the amount ofeach altered reporter signal peptide indicates the amount present in theone or more expression samples of the nucleotide segment that comprisesthe reporter signal peptide from which that altered reporter signalpeptide is derived.

Also disclosed are methods wherein the amount of the nucleotide segmentpresent is proportional to the amount of the altered reporter signalpeptide detected.

Disclosed are methods of detecting expression, the method comprisingdetecting a target altered amino acid segment derived from one or moreexpression samples, wherein the one or more expression samplescollectively comprise a set of nucleic acid molecules, wherein eachnucleic acid molecule comprises a nucleotide segment encoding an aminoacid segment comprising a reporter signal peptide and a protein orpeptide of interest, wherein the amino acid segments have a commonproperty, wherein the common property allows the amino acid segments tobe distinguished and/or separated from molecules lacking the commonproperty, wherein the reporter signal peptides can be altered, whereinalteration of the reporter signal peptides alters the amino acidsegments, wherein the altered form of each amino acid segment can bedistinguished from the altered forms of the other amino acid segments,wherein the target altered amino acid segment is one of the alteredamino acid segments, wherein detection of the target altered amino acidsegment indicates expression of the amino acid segment from which thetarget altered amino acid segment is derived.

Disclosed are methods of detecting expression, the method comprisingdetecting an altered amino acid subsegment derived from one or moreexpression samples, wherein the one or more expression samplescollectively comprise a set of nucleic acid molecules, wherein eachnucleic acid molecule comprises a nucleotide segment encoding an aminoacid segment comprising a reporter signal peptide and a protein orpeptide of interest, wherein the amino acid segments each comprise anamino acid subsegment, wherein each amino acid subsegment comprises aportion of the protein or peptide of interest and all or a portion ofthe reporter signal peptide, wherein the amino acid subsegments have acommon property, wherein the common property allows the amino acidsubsegments to be distinguished and/or separated from molecules lackingthe common property, wherein the reporter signal peptides can bealtered, wherein alteration of the reporter signal peptides alters theamino acid subsegments, wherein the altered form of each amino acidsubsegment can be distinguished from the altered forms of the otheramino acid subsegments, wherein the target altered amino acid subsegmentis one of the altered amino acid subsegments, wherein detection of thetarget altered amino acid subsegment indicates expression of the aminoacid segment from which the target altered amino acid subsegment isderived.

Disclosed are methods of detecting cells, the method comprisingdetecting a target altered reporter signal peptide derived from one ormore cells, wherein the one or more cells collectively comprise a set ofnucleic acid molecules, wherein each nucleic acid molecule comprises anucleotide segment encoding an amino acid segment comprising a reportersignal peptide and a protein or peptide of interest, wherein thereporter signal peptides have a common property, wherein the commonproperty allows the reporter signal peptides to be distinguished and/orseparated from molecules lacking the common property, wherein thereporter signal peptides can be altered, wherein the altered form ofeach reporter signal peptide can be distinguished from the altered formsof the other reporter signal peptides, wherein the target alteredreporter signal peptide is one of the altered reporter signal peptides,wherein detection of the target altered reporter signal peptideindicates the presence of the cell from which the target alteredreporter signal peptide is derived.

Also disclosed are methods wherein each cell is engineered to contain atleast one of the nucleic acid molecules, wherein the reporter signalpeptide of the amino acid segment encoded by the nucleotide segment ofthe nucleic acid molecule in each cell is different.

Also disclosed are methods wherein each cell having a trait of interestcomprises the same reporter signal peptide.

Also disclosed are methods wherein the trait of interest is aheterologous gene.

Also disclosed are methods wherein the heterologous gene comprises thenucleic acid molecule.

Also disclosed are methods wherein the heterologous gene encodes theamino acid segment.

Also disclosed are methods wherein a plurality of different alteredreporter signal peptides are detected, wherein detection of each alteredreporter signal peptide indicates the presence of the cell from whichthat altered reporter signal peptide is derived.

Also disclosed are methods wherein different cells comprise differentnucleic acid molecules, wherein detection of each altered reportersignal peptide indicates the presence of the cell that comprises thenucleic acid molecule that comprises the nucleotide segment encoding theamino acid segment that comprises the reporter signal peptide from whichthat altered reporter signal peptide is derived.

Also disclosed are methods wherein there are a plurality of differentcells, wherein each different cell comprises different nucleic acidmolecules, wherein detection of an altered reporter signal peptideindicates the presence of the cell that comprises the nucleic acidmolecule that comprises the nucleotide segment encoding the amino acidsegment that comprises the reporter signal peptide from which thedetected altered reporter signal peptide is derived.

Disclosed are methods of detecting cell samples, the method comprisingdetecting a target altered reporter signal peptide derived from one ormore cell samples, wherein the one or more cell samples collectivelycomprise a set of nucleic acid molecules, wherein each nucleic acidmolecule comprises a nucleotide segment encoding an amino acid segmentcomprising a reporter signal peptide and a protein or peptide ofinterest, wherein the reporter signal peptides have a common property,wherein the common property allows the reporter signal peptides to bedistinguished and/or separated from molecules lacking the commonproperty, wherein the reporter signal peptides can be altered, whereinthe altered form of each reporter signal peptide can be distinguishedfrom the altered forms of the other reporter signal peptides, whereinthe target altered reporter signal peptide is one of the alteredreporter signal peptides, wherein detection of the target alteredreporter signal peptide indicates the presence of the cell sample fromwhich the target altered reporter signal peptide is derived.

Also disclosed are methods wherein a plurality of different alteredreporter signal peptides are detected, wherein detection of each alteredreporter signal peptide indicates the presence of the cell sample fromwhich that altered reporter signal peptide is derived.

Also disclosed are methods wherein different cell samples comprisedifferent nucleic acid molecules, wherein detection of each alteredreporter signal peptide indicates the presence of the cell sample thatcomprises the nucleic acid molecule that comprises the nucleotidesegment encoding the amino acid segment that comprises the reportersignal peptide from which that altered reporter signal peptide isderived.

Also disclosed are methods wherein there are a plurality of differentcell samples, wherein each different cell sample comprises differentnucleic acid molecules, wherein detection of an altered reporter signalpeptide indicates the presence of the cell sample that comprises thenucleic acid molecule that comprises the nucleotide segment encoding theamino acid segment that comprises the reporter signal peptide from whichthe detected altered reporter signal peptide is derived.

Disclosed are methods of detecting cells, the method comprisingdetecting a target altered reporter signal peptide derived from one ormore cells, wherein the one or more cells collectively comprise a set ofnucleic acid molecules, wherein each nucleic acid molecule comprises anucleotide segment encoding an amino acid segment comprising a reportersignal peptide and a protein or peptide of interest, wherein thereporter signal peptides have a common property, wherein the commonproperty allows the reporter signal peptides to be distinguished and/orseparated from molecules lacking the common property, wherein thereporter signal peptides can be altered, wherein the altered form ofeach reporter signal peptide can be distinguished from the altered formsof the other reporter signal peptides, wherein the target alteredreporter signal peptide is one of the altered reporter signal peptides,wherein detection of the target altered reporter signal peptideindicates the presence of the cell from which the target alteredreporter signal peptide is derived.

Disclosed are methods of detecting cells, the method comprisingdetecting a target altered amino acid segment derived from one or morecells, wherein the one or more cells collectively comprise a set ofnucleic acid molecules, wherein each nucleic acid molecule comprises anucleotide segment encoding an amino acid segment comprising a reportersignal peptide and a protein or peptide of interest, wherein the aminoacid segments have a common property, wherein the common property allowsthe amino acid segments to be distinguished and/or separated frommolecules lacking the common property, wherein the reporter signalpeptides can be altered, wherein alteration of the reporter signalpeptides alters the amino acid segments, wherein the altered form ofeach amino acid segment can be distinguished from the altered forms ofthe other amino acid segments, wherein the target altered amino acidsegment is one of the altered amino acid segments, wherein detection ofthe target altered amino acid segment indicates the presence of the cellfrom which the target altered amino acid segment is derived.

Disclosed are methods of detecting cells, the method comprisingdetecting an altered amino acid subsegment derived from one or morecells, wherein the one or more cells collectively comprise a set ofnucleic acid molecules, wherein each nucleic acid molecule comprises anucleotide segment encoding an amino acid segment comprising a reportersignal peptide and a protein or peptide of interest, wherein the aminoacid segments each comprise an amino acid subsegment, wherein each aminoacid subsegment comprises a portion of the protein or peptide ofinterest and all or a portion of the reporter signal peptide, whereinthe amino acid subsegments have a common property, wherein the commonproperty allows the amino acid subsegments to be distinguished and/orseparated from molecules lacking the common property, wherein thereporter signal peptides can be altered, wherein alteration of thereporter signal peptides alters the amino acid subsegments, wherein thealtered form of each amino acid subsegment can be distinguished from thealtered forms of the other amino acid subsegments, wherein the targetaltered amino acid subsegment is one of the altered amino acidsubsegments, wherein detection of the target altered amino acidsubsegment indicates the presence of the cell from which the targetaltered amino acid subsegment is derived.

Disclosed are methods of detecting organisms, the method comprisingdetecting a target altered reporter signal peptide derived from one ormore organisms, wherein the one or more organisms collectively comprisea set of nucleic acid molecules, wherein each nucleic acid moleculecomprises a nucleotide segment encoding an amino acid segment comprisinga reporter signal peptide and a protein or peptide of interest, whereinthe reporter signal peptides have a common property, wherein the commonproperty allows the reporter signal peptides to be distinguished and/orseparated from molecules lacking the common property, wherein thereporter signal peptides can be altered, wherein the altered form ofeach reporter signal peptide can be distinguished from the altered formsof the other reporter signal peptides, wherein the target alteredreporter signal peptide is one of the altered reporter signal peptides,wherein detection of the target altered reporter signal peptideindicates the presence of the organism from which the target alteredreporter signal peptide is derived.

Also disclosed are methods wherein each organism is engineered tocontain at least one of the nucleic acid molecules, wherein the reportersignal peptide of the amino acid segment encoded by the nucleotidesegment of the nucleic acid molecule in each organism is different.

Also disclosed are methods wherein each organism having a trait ofinterest comprises the same reporter signal peptide.

Also disclosed are methods wherein the trait of interest is a transgeneand methods wherein the transgene gene comprises the nucleic acidmolecule and methods wherein the transgene gene encodes the amino acidsegment.

Also disclosed are methods wherein a plurality of different alteredreporter signal peptides are detected, wherein detection of each alteredreporter signal peptide indicates the presence of the organism fromwhich that altered reporter signal peptide is derived.

Also disclosed are methods wherein different organisms comprisedifferent nucleic acid molecules, wherein detection of each alteredreporter signal peptide indicates the presence of the organism thatcomprises the nucleic acid molecule that comprises the nucleotidesegment encoding the amino acid segment that comprises the reportersignal peptide from which that altered reporter signal peptide isderived.

Also disclosed are methods wherein there are a plurality of differentorganisms, wherein each different organism comprises different nucleicacid molecules, wherein detection of an altered reporter signal peptideindicates the presence of the organism that comprises the nucleic acidmolecule that comprises the nucleotide segment encoding the amino acidsegment that comprises the reporter signal peptide from which thedetected altered reporter signal peptide is derived.

Disclosed are methods of detecting organisms, the method comprisingdetecting a target altered reporter signal peptide derived from one ormore organisms, wherein the one or more organisms collectively comprisea set of nucleic acid molecules, wherein each nucleic acid moleculecomprises a nucleotide segment encoding an amino acid segment comprisinga reporter signal peptide and a protein or peptide of interest, whereinthe reporter signal peptides have a common property, wherein the commonproperty allows the reporter signal peptides to be distinguished and/orseparated from molecules lacking the common property, wherein thereporter signal peptides can be altered, wherein the altered form ofeach reporter signal peptide can be distinguished from the altered formsof the other reporter signal peptides, wherein the target alteredreporter signal peptide is one of the altered reporter signal peptides,wherein detection of the target altered reporter signal peptideindicates the presence of the organism from which the target alteredreporter signal peptide is derived.

Disclosed are methods of detecting organisms, the method comprisingdetecting a target altered amino acid segment derived from one or moreorganisms, wherein the one or more organisms collectively comprise a setof nucleic acid molecules, wherein each nucleic acid molecule comprisesa nucleotide segment encoding an amino acid segment comprising areporter signal peptide and a protein or peptide of interest, whereinthe amino acid segments have a common property, wherein the commonproperty allows the amino acid segments to be distinguished and/orseparated from molecules lacking the common property, wherein thereporter signal peptides can be altered, wherein alteration of thereporter signal peptides alters the amino acid segments, wherein thealtered form of each amino acid segment can be distinguished from thealtered forms of the other amino acid segments, wherein the targetaltered amino acid segment is one of the altered amino acid segments,wherein detection of the target altered amino acid segment indicates thepresence of the organism from which the target altered amino acidsegment is derived.

Disclosed are methods of detecting organisms, the method comprisingdetecting an altered amino acid subsegment derived from one or moreorganisms, wherein the one or more organisms collectively comprise a setof nucleic acid molecules, wherein each nucleic acid molecule comprisesa nucleotide segment encoding an amino acid segment comprising areporter signal peptide and a protein or peptide of interest, whereinthe amino acid segments each comprise an amino acid subsegment, whereineach amino acid subsegment comprises a portion of the protein or peptideof interest and all or a portion of the reporter signal peptide, whereinthe amino acid subsegments have a common property, wherein the commonproperty allows the amino acid subsegments to be distinguished and/orseparated from molecules lacking the common property, wherein thereporter signal peptides can be altered, wherein alteration of thereporter signal peptides alters the amino acid subsegments, wherein thealtered form of each amino acid subsegment can be distinguished from thealtered forms of the other amino acid subsegments, wherein the targetaltered amino acid subsegment is one of the altered amino acidsubsegments, wherein detection of the target altered amino acidsubsegment indicates the presence of the organism from which the targetaltered amino acid subsegment is derived.

Illustrations

The disclosed methods can be further understood by way of the followingillustrations which involve examples of the disclosed methods. Theillustrations are not intended to limit the scope of the method in anyway.

A. Illustration 1: Heavy Isotopes

This illustration makes use of peptide reporter signals having the samemass, that fragment at certain peptide bonds, and that use heavyisotopes to distribute mass differently in different reporter signals.For example, it has been demonstrated, in ion traps, that peptidescontaining arginine will preferentially fragment at the C-termini ofaspartic acid or glutamic acid residues, and, proline containingpeptides will fragment at the N-termini of the proline residues (Qin andChait, Int. J. Mass Spectrom. (Netherlands), 190-191:313-20 (1999)). DP(aspartic acid (D) and proline (P)) amino acid sequences can be used inthe disclosed reporter signals resulting in collisionally inducedfragmentation at the scissile bond between the aspartic acid andproline.

The singly charged ion of an exemplary peptide, AGSLDPAGSLR (SEQ IDNO:2), will fragment between the ‘D’ and ‘P’ in the collision cell ofthe mass spectrometer. Utilizing natural abundance isotopes the singlycharged parent ion will have an average nominal (m/z)=1043 amu, and thepossible resultant daughter ions AGSLD⁺ (amino acids 1-5 of SEQ ID NO:2)and PAGSLR⁺ (amino acids 6-11 of SEQ ID NO:2) have average nominal (m/z)of 461 and 600 amu, respectively. As a practical matter, fragmentationwill typically yield one dominant daughter ion, say PAGSLR⁺ (amino acids6-11 of SEQ ID NO:2) in this case. For this illustration consider onlyone charged daughter from the population of singly charged parent. Notethat, without loss of generality or applicability, the branching ratiointo these daughter ion channels may be other than 100% into the PAGSLR⁺(amino acids 6-11 of SEQ ID NO:2) daughter fragment.

Standard synthetic methods can be utilized to construct such peptides.In this illustration of reporter molecules consider isotopically labeledamino acids (for example, A vs. A*, where A has a CH₃ and A* has a CD₃side chain). There are four possibilities for the synthetic peptide,with their nominal (m/z) indicated in parentheses: AGSLDPAGSLR (1043),A*GSLDPAGSLR (1046), AGSLDPA*GSLR (1046), A*GSLDPA*GSLR (1049) (SEQ IDNO:2). For this example consider the two mono-labeled peptidesA*GSLDPAGSLR, AGSLDPA*GSLR (SEQ ID NO:2), which have a common nominalmass-to-charge of 1046.

As a simple demonstration of a preferred mode of the disclosed methodconsider a solution containing the two synthetic peptides. This solutioncould have been collected following any number of biological experimentsand, in general, because of processing, would contain many additionalcomponents.

The solution containing A*GSLDPAGSLR and AGSLDPA*GSLR (SEQ ID NO:2) ismixed with a suitable matrix solution for performing analysis by massspectrometry. Suitable matrices, including sinapic acid,4-hydroxy-α-cyanocinamic acid or 2,5-dihydroxybenzoic acid, are known inthe art.

The resulting solution is spotted onto the MALDI target and allowed tocrystallize.

The target is inserted into the source of the mass spectrometer.

Utilizing the laser impinging on the sample spot on the MALDI target,many ions are introduced into the first quadrupole, Q0. Among thespecies introduced into Q0 are predominantly singly charged species(A*GSLDPAGSLR⁺, AGSLDPA*GSLR⁺; SEQ ID NO:2), various fragmentation ions,neutral matrix, matrix ions and multimers as known in the art. Neutralparticles will pass out of Q0 without being guided into the secondquadrupole, Q1.

Ions introduced into Q0 are guided into the higher vacuum regioncontaining Q1.

Quadrupole Q1 is set to pass ions with the mass-to-charge ratio of 1046into the third quadrupole, Q2 (recall A*GSLDPAGSLR and AGSLDPA*GSLR (SEQID NO:2) have the same mass-to-charge; “isobaric” in the parlance ofmass spectrometry). Ions with mass-to-charge ratios different from 1046will follow trajectories that do not exit Q1 on the Q1-Q2 axis, and areeffectively discarded. This yields a huge increase in the signal tonoise for the system, on the order of 100-1000 fold improvement oversystems which do not have this mass filtering.

The collision cell surrounding Q2 is filled with a chemically inert gasat an appropriate pressure to cause preferential cleavage of the DPscissile bond of the peptide ions, typically a few milliTorr ofnitrogen. As discussed above, the fragmentation of the singly chargedparent ion is expected to yield predominantly one daughter ion. In thiscase each of the isobaric parents (SEQ ID NO:2) will yield correlated,unique daughters (amino acids 1-5 and 6-11 of SEQ ID NO:2):A*GSLDPAGSLR⁺ → A*GSLD + PAGSLR⁺ (m/z 600) AGSLDPA*GSLR⁺ → AGSLD +PA*GSLR⁺ (m/z 603)

The resolution of the mass spectrometers as discussed here is on theorder of 5000 to 10000, and thus the 3 amu difference is readilyattained at these (m/z).

The ions exiting Q2 enter the time-of-flight (TOF) section of theinstrument. A transient electric field gradient is applied and thepositively charged ions are accelerated toward the reflectron andultimately to the detector. The ions are all accelerated through thesame electric field gradient (the reflectron will compensate for a smallperturbation in this assertion, as is known in the art) and thus willall have the same kinetic energy imparted to them. Because the kineticenergy is the same for all ions, and the masses of the ions aredifferent, the time it takes for the ions to reach the detector will bedifferent: heavier ions will arrive later than lighter ions.

The resulting mass spectrum reflects the relative amount of the twoanalytes (for example, peptides) in the original sample.

This scheme can be extended to more analytes (for example, peptides).The most basic extension for a panel of isobaric detectors based uponthe above peptide, utilizing X/X* differences, would be as shown inTable 2. The asterisk indicates heavy isotope labeled amino acids. Thisset assumes that the non-labeled to labeled mass change{(m/z)_(x*)−(m/z)_(x)} for each residue is the same. For the generalcase where {(m/z)_(x*)−(m/z)_(x)} is not the same for all the residuesthere are more combinations for a given peptide which can be resolved bythe mass spectrometer. The parent molecule is SEQ ID NO:2 and theprimary daughter is amino acids 6-11 of SEQ ID NO:2. TABLE 2 ParentPrimary Daughter A*G*S*L*DPAGSLR PAGSLR AG*S*L*DPA*GSLR PA*GSLRAGS*L*DPA*G*SLR PA*G*SLR AGSL*DPA*G*S*LR PA*G*S*LR AGSLDPA*G*S*L*RPA*G*S*L*R

The synthesis of specific isotope labeled amino acids would facilitaterapidly increased panel size. For example, synthesis of unique alanineswith CH₃, CH₂D, CHD₂, CD₃ side chains could be used to yield asignificant panel size with a small peptide.

This mode of the disclosed method has the desirable property that allthe detected ions originate from a very similar chemical environment(only differing by the location of a few neutrons) and will thus behaveidentically (for all practical purposes) when processed in the MALDIsource and in the collision cell. Of particular note is the case whereone of the isobaric reporter signal molecules is added as a quantitationstandard to the isobaric detector molecules used for the assay.Quantitation of the entire set of detector molecules used in the assayis straightforward and quantitative. For the case where the moleculesare essentially identical except for the isotopic enrichment all theisobars in a set will behave identically through the processing.

B. Illustration 2: Labile Bond, One Daughter Ion.

This illustration makes use of peptide reporter signals having the samemass that fragment at a labile bond, where the labile bond is placed indifferent locations in the different reporter signals. In thisillustration, the parent ion produces a single daughter. An example ofsynthesis of peptides with labile bonds at defined positions betweenamino acids is disclosed by WO 97/11958. Analogous chemistry may beutilized to produce peptides with labile bonds between amino acids foruse in the disclosed method and compositions. For example, consider apair of peptide molecules of the form GSWFSGMCAR (SEQ ID NO:12): PeptideA: GSWFSG#MCAR Peptide B: GSWF#SGMCARwhere the symbol # indicates the location of the labile bond. Note thatthe peptide sequence does not have to be conserved for this method, theonly requirement is that the molecular mass of the peptides be the same.

For simplicity consider a solution containing the two aforementionedsynthetic peptides with labile bonds, A and B. This solution could havebeen collected following any number of biological experiments and, ingeneral, because of processing, would contain many additionalcomponents.

The solution containing A and B is mixed with a suitable matrix solutionfor performing analysis by mass spectrometry. Suitable matrices,including sinapic acid, 4-hydroxy-α-cyanocinamic acid or2,5-dihydroxybenzoic acid, are known in the art.

The resulting solution is spotted onto the MALDI target and allowed tocrystallize.

The target is inserted into the source of the mass spectrometer.

Utilizing the laser impinging on the sample spot on the MALDI target,many ions are introduced into the first quadrupole, Q0. Among thespecies introduced into Q0 are predominantly singly charged species (A⁺,B⁺), various fragmentation ions, neutral matrix, matrix ions andmultimers as known in the art. Neutral particles will pass out of Q0without being guided into Q1.

Ions introduced into Q0 are guided into the higher vacuum regioncontaining Q1.

Quadrupole Q1 is set to pass ions with the mass-to-charge ratio of(m/z)_(A) and (m/z)_(B) (recall (m/z)_(A)=(m/z)_(B); “isobaric” in theparlance of mass spectrometry). Ions with mass-to-charge ratiosdifferent from (m/z)_(A) and (m/z)_(B) will follow trajectories that donot exit Q1 on the Q1-Q2 axis, and are effectively discarded. Thisyields a huge increase in the signal to noise for the system, on theorder of 100-1000 fold improvement over systems which do not have thismass filtering.

The collision cell surrounding Q2 is filled with a chemically inert gasat an appropriate pressure to cause preferential cleavage of the labilebond of the peptide ions A⁺ and B⁺, typically a few milliTorr ofnitrogen. Considering only fragmentation at the labile bond, and theoperation of Q2 in RF only mode, there will be four possible ions whichcan emerge from Q2 into the TOF section. As discussed above, dependingupon the thermodynamics and kinetics, it is common that one of thedaughters for each parent will be more likely to take the charge thanthe other daughter. For the majority of cases there will be onepredominant daughter ion. The primary fragmentation will be (SEQ IDNO:12 into amino acids 1-6 and 7-10 of SEQ ID NO:12 and SEQ ID NO:12into amino acids 1-4 and 5-10 of SEQ ID NO:12): GSWFSG#MCAR⁺ → GSWFSG +MCAR⁺ GSWF#SGMCAR⁺ → GSWF + SGMCAR⁺

The ions exiting Q2 enter the time-of-flight, TOF, section of theinstrument. A transient electric field gradient is applied and thepositively charged ions are accelerated toward the reflectron andultimately to the detector. The ions are all accelerated through thesame electric field gradient (the reflectron will compensate for a smallperturbation in this assertion, as is known in the art) and thus willall have the same kinetic energy imparted to them. Because the kineticenergy is the same for all ions, and the masses of the ions aredifferent, the time it takes for the ions to reach the detector will bedifferent: heavier ions will arrive later than lighter ions.

The resulting mass spectrum shows the relative amount of the tworeporter signals in the original sample.

A standard, with the same mass as the peptides (say GSW#FSGMCAR; SEQ IDNO: 12), could have been added to facilitate quantitative results. Inorder to extract quantitative results the relative efficiencies of theisobaric detector molecule under consideration should be calibrated; astraightforward process.

C. Illustration 3: Labile Bond, Two Daughter Ions.

This illustration makes use of peptide reporter signals having the samemass that fragment at a labile bond, where the labile bond is placed indifferent locations in the different reporter signals. In thisillustration, the parent ion branches into two daughters. Consider thepeptides as described in Illustration 2 (SEQ ID NO:12): Peptide A:GSWFSG#MCAR Peptide B: GSWF#SGMCARwhere the symbol # indicates the location of the labile bond. Note thatthe peptide sequence does not have to be conserved for this method, theonly requirement is that the molecular mass of the reporter moleculepeptides be nominally the same.

For simplicity consider a solution containing the two aforementionedsynthetic peptides with labile bonds, A and B. This solution could havebeen collected following any number of biological experiments and, ingeneral, because of processing, would contain many additionalcomponents.

The solution containing A and B is mixed with a suitable matrix solutionfor performing analysis by mass spectrometry. Suitable matrices,including sinapic acid, 4-hydroxy-α-cyanocinamic acid or2,5-dihydroxybenzoic acid, are known in the art.

The resulting solution is spotted onto the MALDI target and allowed tocrystallize.

The target is inserted into the source of the mass spectrometer.

Utilizing the laser impinging on the sample spot on the MALDI target,many ions are introduced into the first quadrupole, Q0. Among thespecies introduced into Q0 are predominantly singly charged species (A⁺,B⁺), various fragmentation ions, neutral matrix, matrix ions andmultimers as known in the art. Neutral particles will pass out of Q0without being guided into Q1.

Ions introduced into Q0 are guided into the higher vacuum regioncontaining Q1.

Quadrupole Q1 is set to pass ions with the mass-to-charge ratio of(m/z)_(A) and (m/z)_(B) (recall (m/z)_(A)=(m/z)_(B); “isobaric” in theparlance of mass spectrometry). Ions with mass-to-charge ratiosdifferent from (m/z)_(A) and (m/z)_(B) will follow trajectories that donot exit Q1 on the Q1-Q2 axis, and are effectively discarded. Thisyields a huge increase in the signal to noise for the system, on theorder of 100-1000 fold improvement over systems which do not have thismass filtering.

The collision cell surrounding Q2 is filled with a chemically inert gasat an appropriate pressure to cause preferential cleavage of the labilebond of the peptide ions A⁺ and B⁺, typically a few milliTorr ofnitrogen. Considering only fragmentation at the labile bond, and theoperation of Q2 in RF only mode, there will be four possible ions whichcan emerge from Q2 into the TOF section. As discussed above for themajority of cases there will be a predominant daughter ion. Thefragmentation of the population of singly charged parent ions into thedaughter may be as follows (these branching ratios would be empiricallydetermined)(SEQ ID NO:12 into amino acids 1-6 and 7-10 of SEQ ID NO:12and SEQ ID NO:12 into amino acids 1-4 and 5-10 of SEQ ID NO:12):GSWFSG#MCAR⁺ → GSWFSG + MCAR⁺ (A1: 50%) → GSWFSG⁺ + MCAR (A2: 50%)GSWF#SGMCAR⁺ → GSWF + SGMCAR⁺ (B1: 50%) → GSWF⁺ + SGMCAR (B2: 50%)

The branching ratios as noted here would yield a mass spectrum as shownschematically in FIG. 2. The spectrum indicates there is twice as much Bas A in the original sample. In the case of very low pressure in thecollision cell the parent ions will pass through Q2 without fragmenting(FIG. 2A), with gas in the collision cell the peptides will fragment atthe labile bonds (FIG. 2B). Note the correlation (intensities are thesame, and the sum of the masses is equal to the parent ionmass-to-charge) of the A⁺ daughters and the B⁺ daughters.

The ions exiting Q2 enter the time-of-flight, TOF, section of theinstrument. A transient electric field gradient is applied and thepositively charged ions are accelerated toward the reflectron andultimately to the detector. The ions are all accelerated through thesame electric field gradient (the reflectron will compensate for a smallperturbation in this assertion, as is known in the art) and thus willall have the same kinetic energy imparted to them. Because the kineticenergy is the same for all ions, and the masses of the ions aredifferent, the time it takes for the ions to reach the detector will bedifferent: heavier ions will arrive later than lighter ions.

The resulting mass spectrum shows the relative amount of the twoanalytes (for example, peptides) in the original sample. The daughterion signals will be correlated with each other at known branching ratioand known parent ion (m/z), and thus there is increased confidence inthe measurement of the analytes.

A standard, with the same mass as the analytes (say GSW#FSGMCAR; SEQ IDNO:12), could have been added to facilitate quantitative results. Inorder to extract quantitative results the relative efficiencies of theisobars under consideration should be calibrated.

D. Illustration 4: Scissile Bond.

This illustration makes use of peptide reporter signals having the samemass that fragment at certain peptide bonds, where the bond is placed indifferent locations in the different reporter signals. As discussedabove, DP containing amino acid sequence will fragment between theaspartic acid and proline in a collision cell. A set of peptides thatmay be useful for the disclosed method may be: Peptide C: YFMTSGCDPGGR(SEQ ID NO:13) Peptide D: YFMTSGDPCGGR (SEQ ID NO:14) Peptide E:YFMTSDPGCGGR (SEQ ID NO:15) Peptide F: YFMTDPSGCGGR (SEQ ID NO:16)Peptide G: YFMDPTSGCGGR (SEQ ID NO:17)

For simplicity consider a solution containing these synthetic peptides.This solution could have been collected following any number ofbiological experiments and, in general, because of processing wouldcontain many additional components.

The solution containing C, D, E, F, G is mixed with a suitable matrixsolution for performing analysis by mass spectrometry. Suitablematrices, including sinapic acid, 4-hydroxy-α-cyanocinamic acid or2,5-dihydroxybenzoic acid, are known in the art.

The resulting solution is spotted onto the MALDI target and allowed tocrystallize.

The target is inserted into the source of the mass spectrometer.

Utilizing the laser impinging on the spot on the MALDI target, many ionsare introduced into the first quadrupole, Q0. Among the speciesintroduced into Q0 are C⁺, D⁺, E⁺, F⁺, G⁺, various fragmentation ions,matrix ions and multimers as known in the art. Neutral particles willpass out of Q0 without being guided into Q1. Ions introduced into Q0 areguided into the higher vacuum region containing Q1.

Quadrupole Q1 is set to pass ions with the mass-to-charge ratio of(m/z)_(C), (M/z)_(D), (m/z)_(E), (m/z)_(F), (m/z)_(G) (they have thesame molecular weight “isobaric”). Ions with mass-to-charge ratiosdifferent from (m/z)_(C), (m/z)_(D), (m/z)_(E), (m/z)_(F), (m/z)_(G)will follow trajectories which will not exit Q1 on the Q1-Q2 axis, andare effectively discarded. This yields a huge increase in the signal tonoise for the system, on the order of 100-1000 fold improvement oversystems which do not have this mass filtering.

The collision cell surrounding Q2 is filled with a chemically inert gasat an appropriate pressure to cause scission of the D-P bond, typicallya few milliTorr of nitrogen. Considering only fragmentation at the DPbond, and total retention of the charge by the C termini fragments, andthe operation of Q2 in RF only mode, there will be five possible ionswhich can emerge from Q2 into the TOF section. C1⁺: PGGR⁺ (amino acids9-12 of SEQ ID NO:13) D1⁺: PCGGR⁺ (amino acids 8-12 of SEQ ID NO:14)E1⁺: PGCGGR⁺ (amino acids 7-12 of SEQ ID NO:15) F1⁺: PSGCGGR⁺ (aminoacids 6-12 of SEQ ID NO:16) G1⁺: PTSGCGGR⁺ (amino acids 5-12 of SEQ IDNO:17)

The ions exiting Q2 enter the time-of-flight, TOF, section of theinstrument. A transient electric field gradient is applied and thepositively charged ions are accelerated toward the reflectron andultimately to the detector. The ions are all accelerated through thesame electric field gradient (the reflectron will compensate for a smallperturbation in this assertion, as is known in the art) and thus willall have the same kinetic energy imparted to them. Because the kineticenergy is the same for all ions, and the masses of the ions aredifferent, the time it takes for the ions to reach the detector will bedifferent: heavier ions will arrive later than light ions.

The resulting mass spectrum will indicate the relative amount of theanalytes (for example, peptides) in the original sample.

A standard with the same mass as the analytes could have been added tofacilitate quantitative results. In order to extract quantitativeresults the relative efficiencies of molecules under considerationshould be determined to be used in calibration; a straightforwardprocess.

E. Illustration 5: Pre Treatment Direct Readout.

This illustration involves release of the reporter signal from aspecific binding molecule prior to the first quadrupole of theinstrument. The specific binding molecule may be a DNA, a PNA, anantibody, or any other moiety with high specificity and affinity. Thereporter signal is attached to the specific binding molecule through aninteraction which can be selectively broken through the use of, forexample, restriction enzymes, photocleavable nucleotides (WO 00/04036),photocleavable linkages (Olejnik et al., Nucleic Acids Res.,27(23):4626-31 (1999)), and biotin-advidin like interactions (Niemeyeret al., Nucleic Acids Res., 22(25):5530-9 (1994), Sano et al., Science,258(5079):120-2 (1992)).

An exemplary set of constructs might have the general form N_(j)-X_(k),where the nucleotides are indicated by N and are PNA, the amino acidsare indicated by X, the dash indicates the transition from PNA topeptide through a photocleavable linkage, and ‘j’ and ‘k’ areindependent integers. Two members of such an exemplary set are (SEQ IDNO:18; peptide portion): H:ACGGCGACGTGGCTAATC-A*G*S*L*A*G*S*L*DPAGSLAGSLR I:CGAGAGCTAGCTATATGC-AG*S*L*A*G*S*L*DPA*GSLAGSLRwhere the asterisk indicates a heavy amino acid as described inIllustration 1. The PNA will direct specific molecular recognition suchthat ‘H’ will recognize GATTAGCCACGTCGCCGT (SEQ ID NO:19) and ‘I’ willrecognize GCATATAGCTAGCTCTCG (SEQ ID NO:20). Processing in an analogousfashion to the above illustrations, the photocleavable linkage will bebroken by the MALDI laser pulse and the peptide isobar signal moleculeswill be selected by the Q1 mass filter, and one will detect PAGSLAGSLR⁺and PA*GSLAGSLR⁺ (amino acids 10 to 19 of SEQ ID NO:18) for ‘H’ and ‘I’reporter molecules respectively.

Design of DNA-peptide constructs where an internal restriction site isengineered into the DNA strand would enable a DNA specific bindingmolecule and a peptide reporter signal. Endonucleases Hha I, HinP1 I andMnl I are known to have significant single strand activity (NEBcatalog). A prototypical reporter molecule, utilizing Hha I(GCG{circumflex over ( )}C), could have the form (SEQ ID NO:21, DNAportion; SEQ ID NO:18, peptide portion)GACGACGGCGACGTGGCTGCGC-A*G*S*L*A*G*S*L*DPAGSLAGSLRwhere GACGACGGCGACGTGGCT (nucleotides 1 to 18 of SEQ ID NO:21)represents the specific binding molecule, GCGC is the recognition sitefor Hha I, and the dash represents the transition from DNA to peptide.For this mode of the disclosed method, the set of molecules would allshare the underlined sequence adjacent to the transition to the peptide.Pretreatment with Hha I will cleave the all molecules containing GCGCleaving the 3′ cytosine nucleotide attached to the peptide.

In an analogous manner, because one has freedom over the peptidesequence, one can make use of the huge body of literature in the art forspecific cleavage of peptides to specifically cleave the reportermolecule within the peptide moiety. Examples of such specific cleavagesystems include thrombin (cleaves between Arg and Gly), trypsin (cleavesC-terminus of Arg or Lys), endoprotease Glu-C (cleavages C-terminus ofAsp or Glu), and the general classes known as oligopeptidases orendoproteases.

F. Illustration 6: Indirect Readout.

In a preferred embodiment of the disclosed method, a reporter moleculecontaining a decoding tag is used to specifically recognize a codingmolecule. As an example consider a coding molecule which has therecognition sequence as shown for ‘H’ in Illustration 5 (SEQ ID NO:22and SEQ ID NO:23) ACGGCGACGTGGCTAATC-spacer-CGTCATCGTAG

where the specific binding molecule will recognize and associate withGATTAGCCACGTCGCCGT (SEQ ID NO:19), —spacer—is a convenient spacer moietysuch as PEG, and, CGTCATCGTAG (SEQ ID NO:23) represents a coding tag.The reporter molecule is of the form N_(j)-X_(k), where the nucleotidesare indicated by N and are PNA, the amino acids are indicated by X, thedash indicates the transition from PNA to peptide (optionally through acleavable linkage), and ‘j’ and ‘k’ are independent integers. An exampleis (SEQ ID NO:18, peptide portion)CTACGATGACG-A*G*S*L*A*G*S*L*DPAGSLAGSLR

The PNA, which is the decoding tag, will recognize and specificallyassociate with the CGTCATCGTAG (SEQ ID NO:23) coding tag of the codingmolecule ACGGCGACGTGGCTAATC-spacer-CGTCATCGTAG (SEQ ID NO:22 and SEQ IDNO:23)                           GCAGTAGCATC                           |                          A*G*S*L*A*G*S*L*DPAGSLAGSLR (SEQ ID NO:18)

During processing as described above, the reporter molecule ion may beselected by the filter quadrupole, Q1, and read out through the daughterfragments. In the optional case where the link between the PNA and thepeptide may be selectively broken the filter quadrupole, Q1, would betuned to the mass-to-charge of the peptide ion.

A set of molecules for multiplex assay only requires the reportermolecule to have a common mass among the set (or a common mass among theset of peptides, in the case of the selective bond breakage between thePNA and the peptide). A common mass for the reporter molecule is easilyattained simply by utilizing alternate sequence of the PNA preservingthe composition of the PNA (that is, same number of A, C, G, T residuesin all instances) in combination with the peptide isobar detectormolecules as described in Illustration 1.

A clear advantage of this mode of the disclosed method is the ability toseparately optimize the specific binding molecule and the reportersignal of the reporter molecules. A minor constraint on the coding tagof the coding molecule is that among a set the A, C, G, T content mustremain fixed.

G. Illustration 7: Detection of SH2 and SH3 Domains in Proteins of aSingle Sample

This illustration involves detection of individual SH2 and SH3 domainsin particular proteins. The SH2 and SH3 domains of proteins are ofconsiderable interest in the field of proteomics, and of particularrelevance in the field of oncology. Consider a sample containing twoknown proteins that each contain both the SH2 and SH3 domains: humanc-src and v-src. A capture moiety that recognizes these domains (such asan antibody) can be used to select proteins containing these domainsfrom a sample. A pair of such protein sequences is shown below. c-src(NCBI reference GI:11433119; SEQ ID NO:9) MSAIQAAWPS GTE CIAKYNFHGTAEQDLPF CKGDVLTIVA VTKDPNWYKA KNKVGREGII PANYVQKREG VKAGTKLSLMPWFHGKITRE QAERLLYPPE TGLFLVREST NYPGDYTLCV SCDGKVEHYR IMYHASKLSIDEEVYFENLM QLVEHYTSDA DGLCTRLIKP KVMEGTVAAQ DEFYRSGWAL NMKELKLLQTIGKGEFGDVM LGDYRGNKVA VKCIKNDATA QAFLAEASVM TQLRHSNLVQ LLGVIVEEKGGLYIVTEYMA KGSLVDYLRS RGRSVLGGDC LLKFSLDVCE AMEYLEGNNF VHRDLAARNVLVSEDNVAKV SDFGLTKEAS TPRTRASCQS SGQPLRP v-src (NCBI referenceGI:11421794; SEQ ID NO:10) MGSNKSKPKD ASQRRRSLEP AENVHGAGGG AFPASQTPSKPASADGHRGP SAAFAPAAAE PKLFGGFNSS DTVTSPQRAG PLAGGVTTFV ALYDYESRTETDLSFKKGER LQIVNNTEGD WWLAHSLSTG QTGYIPSNYV APSDSIQAEE WYFGKITRRESERLLLNAEN PRGTFLVRES ETTKGAYCLS VSDFDNAKGL NVKHYKIRKL DSGGFYITSRTQFNSLQQLV AYYSKHADGL CHRLTTVCPT SKPQTQGLAK DAWEIPRESL RLEVKLGQGCFGEVWMGTWN GTTRVAIKTL KPGTMSPEAF LQEAQVMKKL RHEKLVQLYA VVSEEPIYIVTEYMSKGSLL DFLKGETGKY LRLPQLVDMA AQIASGMAYV ERMNYVHRDL RAANILVGENLVCKVADFGL ARLIEDNEYT ARQGAKFPIK WTAPEAALYG RFTIKSDVWS FGILLTELTTKGRVPYPGMV NREVLDQVER GYRMPCPPEC PESLHDLMCQ CWRKEPEERP TFEYLQAFLEDYFTSTEPQY QPGENL

The SH3 and SH2 domains are indicated in double underline and singleunderline respectively. Cysteine residues are indicated in bold. Thesecan be labeled by covalent sulfur-sulfur bridges. Tryptic digest of thec-src and v-src proteins results in the fragments shown in Table 3.

Consider a reporter signal of composition CGAGSDPLAGSLR (m/z=1203; SEQID NO:11) and labeling of the cysteine residues of the c-src and v-srcproteins with this reporter signal through formation of covalent bondbetween the sulfur groups of the cysteine of the protein and the sulfurgroups of cysteine of the reporter signal. Table 3 shows the masses ofthe unlabeled, labeled and altered fragments.

In this illustration, the reporter signals are peptides that have beendesigned to have a preferred fragmentation site. Peptides containingarginine will preferentially fragment at the C-termini of aspartic acidor glutamic acid residues, and, proline containing peptides willfragment at the N-termini of the proline residues (Qin and Chait, Int.J. Mass Spectrom. (Netherlands), 190-191:313-20 (1999)). Thus, DP(aspartic acid (D) and proline (P)) amino acid sequences are used in thereporter signals resulting in collisionally induced fragmentation at thescissile bond between the aspartic acid and proline. Labeled mass afterUnlabeled Labeled PLAGSLR mass mass fragment Source Fragment (amu) (amu)loss (amu) c-src, SH3 domain MSAIQAAWPSGTECIAK 1763 2964 2252 c-src, SH3domain YNFHGTAEQDLPFCK 1769 2972 2260 c-src, SH2 domainESTNYPGDYTLCVSCDGK 1951 4353 2929 c-src, SH2 domainLSIDEEVYFENLMQLVEHYTSDADGLCTR 389 1590 878 c-src CIK 362 1563 851 c-srcSVLGGDCLLK 1003 2504 1792 c-src FSLDVCEAMEYLEGNNFVHR 2372 2573 1861c-src ASCQSSGQPLR 1230 2731 2019 v-src, SH2 domain GAYCLSVSDFDNAK 14892690 1978 v-src, SH2 domain HADGLCHR 907 2108 1396 v-srcLTTVCPTSKPQTQGLAK 1772 2973 2261 v-src LGQGCFGEVWMGTWNGTTR 2099 33002588 v-src AANILVGENLVCK 1343 2544 1832 v-src MPCPPECPESLHDLMCQCWR 23747178 4330

Table 3. Fragments resulting from tryptic digest of CGAGSDPLAGSLR (SEQID NO:1) labeled src proteins. The fragments are, from top to bottom,amino acids 1-17 of SEQ ID NO:9, amino acids 18-32 of SEQ ID NO:9, aminoacids 108-125 of SEQ ID NO:9, amino acids 138-166 of SEQ ID NO:9, aminoacids 223-225 of SEQ ID NO:9, amino acids 284-293 of SEQ ID NO:9, aminoacids 294-313 of SEQ ID NO:9, amino acids 346-357 of SEQ ID NO:9, aminoacids 185-198 of SEQ ID NO:10, amino acids 236-243 of SEQ ID NO:10,amino acids 244-260 of SEQ ID NO:10, amino acids 276-294 of SEQ IDNO:10, amino acids 392-404 of SEQ ID NO:10, amino acids 484-503 of SEQID NO:10. Also shown is the resulting mass of the labeled fragment afterloss of the PLAGSLR fragment (amino acids 7-13 of SEQ ID NO:11).

Tryptic digests of the proteins are introduced into a mass spectrometer.Ions corresponding to the masses in the labeled mass column of Table 3are selected and fragmented in the collision cell, subsequently analyzedin the TOF. The collision energy and collision gas density are tunedsuch that the primary fragmentation is the scissile bond betweenaspartic acid (D) and proline (P).

The existence of reporter signals are clearly seen by the parent ionmass-to-charge shift of a multiple of the PLAGSLR (amino acids 7-13 ofSEQ ID NO:1) units upon fragmentation at the scissile bonds, or in somecases (as determined by the molecular dissociation kinetics, dynamicsand thermodynamics) a PLAGSLR⁺ (amino acids 7-13 of SEQ ID NO:11) willbe directly observed. For example, the labeled proteinC(CGAGSDPLAGSLR)IK (amino acids 223-225 of SEQ ID NO:9 and SEQ ID NO:1),shown in row 5 in Table 3, would be selected in the first quadrupole at1563 amu, and would fragment to yield 851 amu (and possibly 712 amu forPLAGSLR⁺; amino acids 7-13 of SEQ ID NO:11). In contrast, unlabeledfragments of the same nominal mass (there are none in this illustration)would be selected by the first quadrupole but would not exhibit the 712amu shift nor the 712 amu peak. This yields an exceptionaldiscrimination against unlabelled fragments. A representation of themass spectrum is shown in FIG. 1.

For an unknown fragment, or to confirm a known fragment, determined tocontain the label, the sequence can be obtained using standard MS/MSpeptide sequencing techniques without further processing.

This illustration demonstrates a simple case of detection of a pair ofknown proteins using the method (via detection of particular fragmentsof the proteins). This method is extensible to a large number ofproteins, known and/or unknown, in a complex mixture. The combinationevent of the parent signal and the fragmentation ion(s) provides anenormous discrimination against the “background”.

If further fractionation is desired automated industrial systems, suchas HPLC or capillary electrophoresis, may be inserted in front of themass spectrometer to increase t the discrimination further.Fractionation systems may be used in tandem arrangement (for example,LC/LC). In the fields of protein discovery and functional genomics,biological fractionation may be employed using interactions of interest,for example a functionally related system such as an affinity partnerfor the SH2 and SH3 domains to capture the families.

H. Illustration 8: Protein Profiling of SH2 and SH3 Domains in Proteinsof a Multiple Samples.

Consider the protein c-src as described in Illustration 7 and itstryptic fragments as described in Table 3.

The temporal protein expression of c-src produced by a stimulus appliedto stable Jurkat T cells (see, for example, Brdicka et al.,Phosphoprotein associated with glycosphingolipid-enriched microdomains(PAG), a novel ubiquitously expressed transmembrane adaptor protein,associates with the protein tyrosine kinase csk and is involved inregulation of T cell activation. J Exp Med, 191:1591-604 (2000)) can befollowed by collecting sample cells at defined time following thestimulus and lysing the cells. The SH2 and SH3 domain containingproteins (including c-src) may be captured at this point in theprocedure. Each lysate is then labeled with a different reporter signalfrom Table 1 and the proteins are digested with trypsin.

Consider the specific example of the c-src tryptic fragment CIK shown inrow 5 in Table 3. For fixed times of 0, 1, 2, 3 and 4 hours the lysatesare labeled with CG*G*G*G*DPGGGGR, CG*G*G*GDPGGGG*R, CG*G*GGDPGGG*G*R,CG*GGGDPGG*G*G*R, and GGGGDPG*G*G*G*R (SEQ ID NO:1), respectively thatwill yield PGGGGR⁺, PGGGG*R⁺, PGGG*G*R⁺, PGG*G*G*R⁺, and PG*G*G*G*R⁺(amino acids 7 to 12 of SEQ ID NO:1) respectively upon collisionalfragmentation. Five time point measurements are obtained in a singlemeasurement by introducing the labeled protein mixture into the massspectrometer, setting the first filter to pass the isobaric set oflabeled proteins (at mass-to-charge corresponding toC(CG*G*G*G*DPGGGGR)IK; SEQ ID NO:1), fragmenting the reporter signal andmeasuring the reporter signals at m/z=499, 500, 501, 502, 503 in orderto detect fragments having the characteristic mass-to-charge ratio foreach of the time points (0, 1, 2, 3 and 4 hours, respectively).

Other labeled proteins of interest are selected with the filter andquantitated in similar fashion.

I. Illustration 9: Protein Fragment Detection with Reporter SignalCalibrators

This illustration involves detection of protein fragments using reportersignal calibrators.

1. A suspension containing 1000 cells is centrifuged to get a cellpellet.

2. The cells are lysed using detergent.

3. The lysate is digested with trypsin.

4. Optionally, the protein digest is oxidized with hydrogen peroxide orderivatized with acetylacetone.

5. The material placed in a tandem mass spectrometer, ionized, selectedby a mass-to-charge filter, fragmented, mass analyzed, and detected, inorder to measure the signals from unique fragile tryptic peptides andthe corresponding reporter signal calibrator standard designed for eachunique tryptic peptide.

6. Mass spectrometry detection is repeated with different, specificfiltering settings for 50 different peptide mass/charge ratios suitablefor each signature tryptic peptide and its corresponding reporter signalcalibrator peptide.

7. Data is collected as a catalog of 50×2 independent measurements,constituting the peptide signature.

J. Illustration 10: Reporter Signal Fusions, Expressed From PlasmidVectors, With Epitope Tags

This illustration provides an example of, and an example of the use of,a set of simple expression vectors encoding an amino acid segment thatincludes an epitope tag that is the same in all the vectors, and thatincludes a reporter signal peptide that is different in all the vectorsof the set of vectors. The reporter signal peptides can be cleaved fromthe amino acid segment with trypsin. All of the reporter signal peptideshave the same mass-to-charge ratio, but, when fragmented, producefragments that have different mass-to-charge ratios.

1. A set of different DNA plasmid vectors is constructed containing thefollowing elements:

-   -   (a) a common origin of replication and a common antibiotic        selectable marker,    -   (b) an inducible promoter (for this illustration, the promoter        could be the same for all plasmids or different for each        plasmid), and    -   (c) a nucleic acid segment encoding an amino acid segment (the        reporter signal fusion) where the amino acid segment includes:        -   (1) a protein of interest to be expressed under the control            of (that is, operably linked to) the promoter (for this            illustration, the protein could be different for each            plasmid or could be the same for all plasmids),        -   (2) a common epitope tag, such as a flag peptide, and        -   (3) a reporter signal peptide that can be released from the            amino acid segment upon trypsin digestion (for this            illustration, each plasmid encodes a different reporter            signal peptide where each reporter signal peptide belongs to            the same isobaric set of reporter signal peptides).

2. Each plasmid vector is introduced individually intotransformation-competent cells.

3. Transformed cells are grown under the antibiotic selection.

4. The inducible promoter is induced by its appropriate activatorcompound.

5. The expressed amino acid segments (that is, reporter signal fusions)are measured as follows:

-   -   (a) the cells, harboring different expression vectors, are mixed        in a single vessel,    -   (b) the mixture of cells is lysed to release all proteins,    -   (c) an antibody specific for the epitope tag is used to purify        (separate) the reporter signal fusion(s) from the lysate,    -   (d) the epitope tag-purified reporter signal fusion is digested        with trypsin,    -   (e) the tryptic peptides are combined with matrix and analyzed        by MALDI-tandem mass spectrometry where the amount of each        different reporter signal is measured.        K Illustration 11: Reporter Signal Fusions, Expressed From        Plasmid Vectors, With Cis-Cleavable Linkage

This illustration provides an example of, and an example of the use of,a set of expression vectors encoding reporter signal fusions with apeptide-release mechanism based on activatable self-cleavage proteolyticactivity of an intein, or any suitable cis-acting protease. Theproteolytic activity serves to control the release of the reportersignal peptide present in the each of the reporter signal fusions.

1. A set of different DNA plasmid vectors is constructed harboring thefollowing sequence elements:

-   -   (a) an origin of replication and an antibiotic selectable        marker,    -   (b) an inducible promoter, wherein the promoter may be the same        for all plasmids, or different for each plasmid,    -   (c) a nucleic acid segment encoding an amino acid segment (the        reporter signal fusion), to be expressed under the direction of        the promoter, where the amino acid segment includes:        -   (1) a protein of interest, wherein the protein could be            different for each plasmid, or could be the same for all            plasmids,        -   (2) an intein protein domain located such as to be able to            catalyze release of the reporter signal peptide by a            cis-cleavage reaction.        -   (2) a reporter signal peptide belonging to a specific            isobaric set of reporter signal peptides, wherein each            plasmid encodes a different member of the isobaric set of            reporter signal peptides.

2. The plasmid vector is introduced into transformation-competent cells.

3. Transformed cells are grown under the antibiotic selection.

4. The inducible promoter is induced by its appropriate activatorcompound.

5. The expressed reporter signal fusion is measured as follows:

-   -   (a) the cells are lysed to release internal proteins,    -   (b) DTT is added to activate the intein self-cleavage activity        (Chong et al. (1998) Utilizing the C-terminal cleavage activity        of a protein splicing element to purify recombinant proteins in        a single chromatographic step. Nucleic Acids Res 26:5109-5115),    -   (c) the released peptides are combined with matrix and analyzed        by MALDI-tandem mass spectrometry where the amount of each        different reporter signal is measured.        L. Illustration 12: Reporter Signal Fusions, Expressed from BAC        Vectors, with Epitope Tags

This illustration provides an example of, and an example of the use of,a set of mammalian BAC expression vectors with recombinase sites capableof driving integration in specific gene loci.

1. A set of different BAC vectors derived from pEYMT (Hong et al. (2001)Development of two bacterial artificial chromosome shuttle vectors for arecombination-based cloning and regulated expression of large genes inmammalian cells. Analytical Biochemistry 291:142-148) is constructed.These vectors are capable of shuttling between bacteria, yeast andmammalian cells. The vectors have the following features:

-   -   (a) a common promoter,    -   (b) a nucleic acid segment encoding an amino acid segment (the        reporter signal fusion), to be expressed under the direction of        the promoter, where the amino acid segment includes:        -   (1) one of a set of proteins of interest, wherein the            protein coding sequence is different for each BAC, or,            alternatively, the protein coding sequence is the same for            all BACs, but the BACs then are made different by the use of            a different promoter in each BAC,        -   (2) an epitope tag, such as the flag peptide,        -   (3) a reporter signal peptide belonging to a specific            isobaric set of reporter signal peptides, whereby the            reporter signal fusion is tagged with the epitope tag and a            unique reporter signal peptide, whereby the reporter signal            peptide may be released by trypsin digestion.

2. Each of the BAC vectors is introduced individually into mouseembryonic stem cells, to achieve integration in genomic DNA. Thetransformed ES cells are introduced into an embryo, to generate achimeric animal, containing ES cells in the germline. The progeny ofthese mice are screened to identify transgenic mice that harbor theintegrated reporter signal fusion construct.

3. Tissue is obtained from each transgenic animal, and equal amounts oftissue from several animals is mixed.

4. The mixture of tissues is lysed to release proteins.

5. An antibody specific for the epitope tag (for example, anti-flagantibody) is used to purify the reporter signal fusions.

6. The flag-purified proteins are digested with trypsin.

7. The tryptic peptides are combined with matrix and analyzed byMALDI-tandem mass spectrometry where the amount of each differentreporter signal is measured.

M. Illustration 13: Reporter Signal Fusions, Expressed from PlantVectors, with Epitope Tags and Recombinase Sites

This illustration provides an example of, and an example of the use of,a set of plant expression vectors with two directly oriented lox sitesites capable of driving integration in a specific recipient gene locus(slightly modified from Vergunst et al. (1998) Site-specific integrationof Agrobacterium T-DNA in Arabidopsis thaliana mediated by Crerecombinase. Nucleic Acids Res 26:2729-2734).

1. A set of different Agrobacterium T-DNA vectors is constructedharboring the following sequence elements:

-   -   (a) A Floxed T-DNA recombination cassette, without a promoter        (Vergunst et al. (1998) Site-specific integration of        Agrobacterium T-DNA in Arabidopsis thaliana mediated by Cre        recombinase. Nucleic Acids Res 26:2729-2734), designed to be        integrated in the genome of a recipient plant by Cre        recombinase-driven integration, with the cassette comprising a        nucleic acid segment encoding an amino acid segment (the        reporter signal fusion), to be expressed under the direction of        the promoter, where the amino acid segment includes:        -   (1) a coding sequence for a protein of interest, wherein the            protein could be different for each T-DNA, or could be the            same for all T-DNAs,        -   (2) an epitope tag, such as the flag peptide,        -   (3) a reporter signal peptide belonging to a specific            isobaric set of reporter signal peptides.

2. The T-DNA plasmid vector is introduced into recipient plants, suchplants harboring a chimeric promoter-lox-Cre gene, under the control ofa chemically inducible promoter (Kunkel et al. (1999) Inducibleisopentenyl transferase as a high-efficiency marker for planttransformation. Nature Biotechnology 17:916-919), designed to receivethe recombinant protein cassette of the integrative vector by Cre-drivenrecombination. As in the original design of Vergunst and co-workers(1998), site-specific integration simultaneously leads to loss ofCre-expression, making the insertion event irreversible.

3. The expressed, integrated reporter signal fusion is generated underthe direction of the chemically inducible promoter present in front ofthe integrated gene. Expression is measured as follows:

-   -   (a) tissue is obtained from each plant, and equal amounts of        tissue from several plants is mixed,    -   (b) the mixture of tissues is lysed to release proteins,    -   (c) an antibody specific for the epitope tag (i.e., anti-flag)        is used to purify the reporter signal fusions,    -   (d) the flag-purified proteins are digested with trypsin,    -   (e) the tryptic peptides are combined with matrix and analyzed        by MALDI-tandem mass spectrometry where the amount of each        different reporter signal is measured.        N. Illustration 14: Kit Including Vectors Encoding Reporter        Signal Fusions

This illustration provides an example of a kit comprising a set of 2 ormore vectors encoding reporter signal fusions. The kit comprises two ormore expression vectors, wherein each vector expresses a differentreporter signal fusion, wherein all reporter signal peptides in thereporter signal fusions belong to single isobaric set. The kit also cancontain reagents needed for use of the vectors, such as

-   -   (a) Reagents for transformation,    -   (b) Reagents for inducing release of reporter signal peptides        from reporter signal fusions, and    -   (c) Reagents for performing mass spectral analysis, such as a        matrix optimized for analysis of reporter signal peptides.        O. Illustration 15: Reporter Signal Fusions Encoded by PCR        Products

This illustration provides an example of, and an example of the use of,a set of reporter signal fusions designed for expression in a rabbitreticulocyte cell-free system, where the DNA encoding the reportersignal fusion is generated by PCR.

1. PCR primers are designed to amplify a protein sequence of interest,whereby the one of the PCR primers contains a T7 RNA polymerasepromoter, and a Kozak translational initiation sequence, positionedcorrectly in relation to the AUG start codon. The PCR primers alsocontain sequences coding for a reporter signal peptide, which may beplaced at the amino terminus or at the carboxyl terminus of the proteinof interest (thus forming a reporter signal fusion). Each reportersignal peptide is designed such as to cleavable from the protein bytrypsin digestion.

2. The artificial gene is amplified by PCR, to generate sufficient DNA.

3. The DNA generated by PCR is transcribed in vitro using T7 RNApolymerase.

4. The solution containing the transcribed DNA is added to a rabbitreticulocyte in vitro translation system, to generate the reportersignal fusion product.

5. The in vitro synthesized reporter signal fusion is used with orwithout purification.

6. The process is repeated for other variants of the protein ofinterest. In a typical application, as many as 128 different proteinvariants may be generated by in vitro transcription/translation.

P. Illustration 16: Reporter Signal Fusions Encoded by PCR Products

This illustration provides an example of, and an example of the use of,a set of reporter signal fusions encoding by nucleic acid moleculesdesigned for expression in an E. coli coupled transcription/translationsystem.

1. PCR primers are designed to amplify a protein sequence of interest,whereby the one of the PCR primers contains a T7 RNA polymerasepromoter, and a Shine-Dalgarno translational initiation sequence,positioned correctly in relation to the AUG start codon. The PCR primersalso contain sequences coding for a reporter signal peptide, which maybe placed at the amino terminus or at the carboxyl terminus of theprotein of interest (thus forming a reporter signal fusion). Eachreporter signal peptide is designed such as to cleavable from theprotein by trypsin digestion.

2. The artificial gene is amplified by PCR, to generate sufficient DNAfor use in a coupled in vitro transcription/translation system.

3. The DNA generated by PCR is incubated in the in vitro coupledtranscription/translation system, to generate the reporter signal fusionproduct.

4. The in vitro synthesized reporter signal fusion is used with orwithout purification.

5. The process is repeated for other variants of the protein ofinterest. In a typical application, as many as 128 different proteinvariants may be generated by in vitro transcription/translation.

Q. Illustration 17: Reporter Signal Fusions Expressed in Yeast Cells

This illustration provides an example of, and an example of the use of,a set of 32 yeast strains, each strain harboring a single reportersignal fusion.

A yeast strain (Saccharomyces cerevisiae) is constructed, usinghomologous recombination targeted to a non-essential gene, whereby afusion of a candidate therapeutic protein and a reporter signal peptide(belonging to a set of 32 isobaric reporter signal peptides) is placedunder the control of a galactose-responsive promoter. Another 31 similaryeast strains are constructed, using the same yeast promoter, wherebythe only other difference in the DNA sequence coding for the reportersignal fusion is the use of codons designed to generate one of 31different reporter signal peptides, completing a set of 32 differentpromoters and an isobaric set of 32 distinct reporter signal peptides.The yeast strains may be used for any assay where the reporter signalpeptides (and/or reporter signal fusions) serve as reporters for theexpression of the protein fused to the reporter signal peptide, which inthis case is a candidate therapeutic protein.

R. Illustration 18: Reporter Signal Fusions Expressed in Mouse Cells

This illustration provides an example of, and an example of the use of,a set of 32 mouse cell lines, each cell line harboring a single reportersignal fusion.

A mouse cell line is constructed, using an SV40 vector system, whereby afusion of a candidate therapeutic protein and a reporter signal peptide(belonging to a set of 32 isobaric reporter signal peptides) is placedunder the control of a cytokine promoter. Another 31 similar cell linesare constructed, using 31 different cytokine promoters, whereby the onlyother difference in the DNA sequence coding for the reporter signalfusion is the use of codons designed to generate one of 31 differentreporter signal peptides, completing a set of 32 different promoters andan isobaric set of 32 distinct reporter signal peptides. The cell linesmay be used for any assay where the reporter signal peptides (and/orreporter signal fusions) serve as reporters for the expression of theprotein fused to the reporter signal peptide, which in this case is acandidate therapeutic protein.

S. Illustration 19: Reporter Signal Fusions Expressed Using Promoter ofInterest

This illustration provides an example of the use of cells that harborsingle fusions, as part of a cytokine-STAT5a-responsive promoter. Thissystem can provide a comparison of reporter signal peptides versus a GFPinternal standard.

1. An experiment is performed using a set of 96 different reportersignal peptides belonging to a unique mass set (that is, an isobaricset), where each cell line harbors a nucleic acid construct encoding asingle reporter signal fusion. The fusion construct contains acytokine-responsive promoter for the CIS 1 protein, which is activatedthrough a STAT5 response (Masumoto et al. (1999) Suppression of STAT5functions in liver, mammary glands, and T cells in cytokine-inducible,SH2-containing protein 1 transgenic mice. Mol Cell Biol 19:6396-6407), aGFP-encoding sequence and a sequence encoding a reporter signal peptidefused to the GFP.

2. After treatment of 38,400 cell cultures for 6 hours with a set of38,400 different cytokine-mimic drug candidates from a combinatorialdrug library, a subset of 3,840 cell cultures are sampled to obtainvalues for GFP fluorescence.

3. The cells are pooled in groups of 96.

4. The mixture of cells is lysed to release the GFP fusion proteins.

5. The lysate is digested with trypsin.

6. The tryptic peptides are combined with matrix and analyzed byMALDI-tandem mass spectrometry where the amount of each differentreporter signal is measured.

The readout speed of an expensive mass spectrometer is often therate-limiting factor in proteomic analysis. A key feature of this methodis the ability to pool sets of 96 treated cell samples prior toimmunoprofiling based on mass spectrometry of reporter signal peptides.A total of 38,400 cell cultures are treated, each with a different drug.The use of pooling different cells harboring one of 96 differentreporter signal peptides permits the 38,400 cultures to be analyzed as400 pooled samples.

The GFP fluorescence values obtained in step 2 for a subset of thesamples (3,840 out of 38,400) are compared to the data obtained by massspectrometry analysis of the reporter signal peptides from the samesamples. A good correlation between the fluorescence values and thevalues obtained by mass spectrometry constitutes a control for thefunction and utility of an reporter signal fusion-tagged cell line.

T. Illustration 20: Multiple Reporter Signal Fusions Expressed in a Cell

This illustration provides an example of a method for expressionprofiling of 32 different reporter signal fusions, utilizing cells thatharbor multiple reporter signal fusions.

An experiment is performed using a set of 32 different reporter signalpeptides belonging to a unique mass set (that is, they are isobaric),where each cell line harbors 32 different reporter signal fusions thatare expressed independently, under the control of different promoters.Monitoring the expression of these 32 different proteins serves as ameasure of drug toxicity.

Cell cultures are analyzed one at a time:

-   -   (a) the cells are lysed to release proteins,    -   (b) the lysate is digested with trypsin.    -   (c) the tryptic peptides are combined with matrix and analyzed        by MALDI-tandem mass spectrometry where the amount of each        different reporter signal is measured.        U. Illustration 21: Multiple Reporter Signal Fusions Expressed        in a Mammalian Cell Line

This illustration provides an example of, and an example of the use of,a mammalian cell line, designed for use as a microencapsulated producerfor heterologous protein delivery, the cell line harboring 12 reportersignal fusions.

A mammalian cell line is constructed, using a BAC homologousrecombination vector system (Hong et al. (2001) Development of twobacterial artificial chromosome shuttle vectors for arecombination-based cloning and regulated expression of large genes inmammalian cells. Analytical Biochemistry 291:142-148), whereby a fusionof two candidate therapeutic proteins and two isobaric reporter signalpeptides (belonging to a set of 12 isobaric reporter signal peptides) isplaced under the control of a tetracycline promoter. Another 10 fusionsin 10 genes, coding for different secretory proteins, are constructed inthe same cell line, under the control of their own native promoters,whereby the only other difference in the DNA sequence coding for thefusion peptides is the use of codons designed to generate one of 10different reporter signal peptides. This completes a set of 12 differentgenes and 12 distinct reporter signal peptides.

The cell line is microencapsulated, and used for heterologous proteindelivery in an animal host, or a human patient, where secretion of thetherapeutic proteins is induced by tetracycline.

An immunoassay is performed, where specific antibodies are used tocapture the 12 reporter signal fusions of interest, whereby the reportersignal peptides (and/or reporter signal fusions) serve as reporters forthe expression of each protein fused to one reporter signal peptide,including the two candidate therapeutic proteins. One may thus measurethe response of the microencapsulated cells to tetracycline induction,and, simultaneously, the production of other secretory proteins by themicroencapsulated cells.

This precise monitoring of protein expression by the microencapsulatedcells, using reporter signal fusions, serves to accelerate the dosageoptimization, and results in increased therapeutic safety control. Amongthe additional secretory proteins monitored by the method one mayinclude cytokines or other proteins with mitogenic potential.

V. Illustration 22: Multiple Reporter Signal Fusions Expressed inMultiple Cell Lines

This illustration provides an example of, and an example of the use of,a set of six different human cell lines, each cell line harboring tendifferent reporter signal fusions, whereby all reporter signal peptidesbelong to the same isobaric set.

Six cell lines are derived from adult stem cells, where each cell lineis representative of a different major human haplotype, defined byunique SNP combinations, whereby each of the six haplotypes isrepresentative of an important pharmacogenomic drug response subset ofthe human population for beta(2)-adrenergic receptor (Drysdale et al.(2000) Complex promoter and coding region beta 2-adrenergic receptorhaplotypes alter receptor expression and predict in vivo responsiveness.Proc Natl Acad Sci USA. 97:10483-10488).

A total of six mammalian cell lines are constructed, using a BAChomologous recombination vector system (Hong et al. (2001) Developmentof two bacterial artificial chromosome shuttle vectors for arecombination-based cloning and regulated expression of large genes inmammalian cells. Analytical Biochemistry 291:142-148), whereby a set often fusions in ten different genes, coding for different signaltransduction proteins, are constructed in same cell line, under thecontrol of their own native promoters, whereby the only other differencein the DNA sequence coding for the fusion peptides is the use of codonsdesigned to generate only one of ten different reporter signal peptides.This completes a set of ten different genes and ten corresponding,distinct reporter signal fusions for each cell line, whereby each cellline represents a major haplotype of the human beta(2)-adrenergicreceptor, and thus sixty different reporter signal fusions are presentin the combined set of all six cell lines.

The cell lines may be used for any assay where the set of sixty reportersignal peptides serve as reporters for the expression of the specificproteins fused to each reporter signal peptide in each cell line.

W. Illustration 23: Multiple Reporter Signal Fusions Expressed inMultiple Human Cell Lines

This illustration provides an example of, and an example of the use of,a set of six cell lines, where each cell line is representative of ahuman haplotype, and each cell line harbors multiple reporter signalfusions.

1. An experiment is performed using a set of sixty different reportersignal peptides belonging to a unique mass set (that is, they areisobaric), where each cell line harbors ten reporter signal fusions. Theobjective of the experiment is to measure the response of the cells to adrug.

2. The cells are pooled in groups of six haplotypes.

3. The mixture of cells is lysed to release proteins.

4. The lysate is digested with trypsin.

5. The tryptic peptides are combined with matrix and analyzed byMALDI-tandem mass spectrometry where the amount of each differentreporter signal is measured.

The readout speed of an expensive mass spectrometer is often therate-limiting factor in proteomic analysis. A key feature of this methodis the ability to pool sets of six treated cell samples prior toimmunoprofiling based on mass spectrometry of reporter signal peptides.A total of 2,400 cell cultures are treated, each with a different drug.The use of pooling of six different cell lines permits the 2,400cultures to be analyzed as 400 pooled samples. All 400 samples aredeposited on the plate of a mass spectrometer, and analyzed by tandemmass spectrometry. The information for each laser shot consists of theexpression levels of sixty different reporter signal fusions.

X. Illustration 24: Kit of Reporter Signal Fusion-Labeled Human CellLines

This illustration provides an example of, and an example of the use of,a kit comprising six cell lines, where each cell line is representativeof a major human haplotype, and each cell line harbors multiple reportersignal fusions. The kit includes the cell lines, and a set of reportersignal peptide controls designed to be used in experiments that areperformed using a set of sixty different reporter signal peptidesbelonging to a unique mass set, where each cell line harbors tenreporter signal fusions. The objective of the experiment is to theresponse of the cells to different drugs.

Y. Illustration 25: Reporter Signal Fusion-Labeled Flies

This illustration provides an example of, and an example of the use of,a transgenic fruit fly harboring reporter signal fusions.

A recombinant fly of the species Drosophila melanogaster is constructed,using homologous recombination (Rong & Golic (2001) A targeted geneknockout in Drosophila. Genetics 157:1307-1312), so that a total of 16genes are modified by addition of sequence encoding reporter signalfusions belonging to a unique mass set (that is, they are isobaric). The16 recombinant proteins are chosen on the basis of their known functionat various levels of different signal transduction pathways, such asras, myc, etc. The fusion is located at either the carboxyl-terminus orthe amino-terminus of each of the proteins, and may optionally bepreceded by an epitope tag, such as the flag epitope.

The flies are used for an experiment in which a new genotype isgenerated by transformation with P-elements harboring members of arecombinant protein library. The objective of performing thetransformation is to observe the phenotypes generated by differentprotein sequences present in the recombinant library.

After transformation, individual flies are processed to extractproteins, the proteins are digested with trypsin, and the reportersignal peptides derived from reporter signal fusions are analyzed bydesorption-ionization using a nanostructured silicon film (Hayes et al.(2001) Desorption-ionization mass spectrometry using depositednanostructured silicon films. Anal. Chem. 73:1292-1295), coupled withcollision-induced fragmentation tandem mass spectrometric analysis. Thereporter signal peptide profile generates a representation of therelative abundance of the reporter signal fusions in the fly.

Z. Illustration 26: Reporter Signal Fusion-Labeled Mice

This illustration provides an example of, and an example of the use of,transgenic mice harboring reporter signal fusions for signaltransduction pathway analysis.

A recombinant mouse of the species Mus musculus is constructed, usinghomologous recombination in embryonic stem (ES) cells, (Templeton et al.(1997) Efficient gene targeting in mouse embryonic stem cells. GeneTherapy 4:700-709), so that a total of 12 genes are modified by additionof reporter signal fusions belonging to a unique mass set (that is, theyare isobaric). The gene fusions are designed by adding the reportersignal peptide at either the amino terminus or the carboxyl-terminus ofeach of the recombinant proteins of interest. The 12 recombinantproteins are chosen on the basis of their known key functions at variouslevels of different signal transduction pathways, such as ras, myc, wnt,etc. Some of the fusions may optionally contain an epitope tag, such asthe flag epitope, or a GFP fusion. Some of the fusions may involve mouseproteins of unknown function.

Most of the recombinant reporter signal fusions are placed under theirnormal mouse promoter, while one or a few of the recombinant reportersignal fusions may be under the control of a heterologous promoter, totest a certain experimental hypothesis. For example, an experimentalrecombinant reporter signal fusion may consist of an interleukin-6coding sequence, fused to an reporter signal peptide, under the(inappropriate) control of the interleukin-2 promoter.

Mice with normal promoters, as well as the mice with an experimentalheterologous promoters linked to reporter signal fusions, are used in aseries of experiment in which tumors are induced by a chemical mutagen(2-azoxymethane). After tumors appear, the mice are treated withdifferent candidate anti-tumor drugs.

At different times after drug treatment, tumors are dissected fromindividual mice, and the tumor tissue is processed to extract proteins.The proteins are digested with trypsin, and the reporter signal peptidesderived from reporter signal fusions are analyzed bydesorption-ionization using a nanostructured silicon film (Hayes et al.(2001) Desorption-ionization mass spectrometry using depositednanostructured silicon films. Anal. Chem. 73:1292-1295), coupled withcollision-induced fragmentation tandem mass spectrometric analysis. Thereporter signal peptide profile generates a representation of therelative abundance of the 12 reporter signal fusions, and this profileserves as an informative measure of multiple pathway responses to theantitumor drug in a normal mouse, or in a mouse with experimentalheterologous promoter constructs. The profiles may also provideinformation regarding the expression of proteins of unknown function.

AA. Illustration 27: Reporter Signal Fusion-Labeled Mice

This illustration provides an example of, and an example of the use of,transgenic mice harboring reporter signal fusions for studyinginflammatory responses and Cyclooxygenase 2 (Cox-2) promoter mutants.

A recombinant mouse of the species Mus musculus is constructed, usinghomologous recombination in embryonic stem (ES) cells, (Templeton et al.(1997) Efficient gene targeting in mouse embryonic stem cells. GeneTherapy 4:700-709), so that a total of ten genes are modified byaddition of reporter signal fusions belonging to a unique mass set. Thegene fusions are designed by adding the reporter signal peptide ateither the amino terminus or the carboxyl-terminus of each of therecombinant proteins of interest. The ten recombinant proteins arechosen on the basis of their known function at various levels of tissueinflammatory responses (such as Cox-2, etc). Some of the fusions mayoptionally contain an epitope tag, such as the flag epitope, or a GFPfusion. Some of the fusions may involve mouse proteins of unknownfunction, but which are suspected to have a role in inflammation.

Most of the recombinant reporter signal fusions are placed under theirnormal mouse promoter, while one or a few of the recombinant reportersignal fusions may be under the control of a mutant promoter, to test acertain experimental hypothesis. For example, an experimentalrecombinant reporter signal fusion may consist of a Cox-2 codingsequence, fused to an reporter signal peptide, under the control of areduced transcriptional response Cox-2 mutant promoter.

Mice with normal promoters, as well as the mice with an experimentalmutant promoters linked to reporter signal fusions, are used in a seriesof experiments in which colonic inflammation and colitis is induced byDextran sulfate, an then the mice are treated with anti-inflammatorydrugs.

At different times after drug treatment, colons are dissected fromindividual mice, and the tissue is processed to extract proteins. Theproteins are digested with trypsin, and the reporter signal peptidesderived from reporter signal fusions are analyzed bydesorption-ionization using a nanostructured silicon film (Hayes et al.(2001) Desorption-ionization mass spectrometry using depositednanostructured silicon films. Anal. Chem. 73:1292-1295), coupled withcollision-induced fragmentation tandem mass spectrometric analysis. Thereporter signal peptide profile generates a representation of therelative abundance of the ten reporter signal fusions, and this profileserves as an informative measure of multiple pathway responses to theanti-inflammatory drug in the colon of a normal mouse with colitis, orin a mouse with experimental mutant Cox-2 promoter constructs. Theprofiles may also provide information regarding the expression ofproteins of unknown function.

BB. Illustration 28: Multiple Samples Labeled with Different ReporterSignals

This illustration is an example of multiple sample labeling usingreporter signals where each sample is labeled with a different reportersignal.

This illustration involves the use of 384 antibody mini-columns, inorder to generate a profile of 384 different protein antigens.Antibodies are covalently coupled to agarose beads using standardwater-soluble carbodiimide chemistry. (March et al. (1974) A simplifiedmethod for cyanogen bromide activation of agarose for affinitychromatography. Anal Biochem. 60:149-152). The capacity of a small (75microliter) affinity column with one antibody is equal to approximately2×10¹¹ molecules of each protein. If multiplexing of reporter signals is256×, the maximum protein binding capacity will be approximately 10⁸molecules. The dynamic range of the assay will thus be 10,000 to100,000,000 molecules of protein.

1. Prepare 4 sets of 64 reporter signals (for a total of 256), each ofthe four sets having the same mass.

2. Label each of 256 cell preps by covalent coupling of a uniquereporter signal, using standard heterobifunctional reagents such as SATAand SSCP (Pierce Chemicals). Preferred chemistry for this purpose is theuse of Sulfo-LS-SPDP (cat #21650 from Pierce; Uto et al., Determinationof urinary Tamm-Horsfall protein by ELISA using a maleimide method forenzyme-antibody conjugation, J. Immunol. Methods, 138:87-94 (1991).

3. Associate with affinity column on microtip containing specificantibody.

4. Repeat for 384 antibodies. That is, associate all of the labeled cellpreps to each of the 384 columns.

5. Elute from column using photocleavable reportersignal-release-chemistry (Innovachem, Tucson, Ariz.; see, for example,Harth-fritschy and Cantacuzene, Pept. Res. 50:415 (1997)).

6. The reporter signals are combined with matrix and analyzed byMALDI-tandem mass spectrometry where the amount of each differentreporter signal is measured, using 4 successive mass-to-charge settings(4×64), one for the mass of each of the four sets.

The number of data points will be 256×384=98,304. Ten runs per day wouldprovide 980,000 data points. This method is easily scalable to 384×10antibodies=3840 antibodies. For 3840 antibodies, 10 runs per day wouldgive 9,830,400 data points per day.

CC. Illustration 29: Multiple Samples Labeled with Different ReporterSignals

This illustration is an example of multiple sample labeling usingreporter signals where each sample is labeled with a different reportersignal. The samples are labeled via a DNA coding tag intermediate.

This illustration involves the use of 384 antibody mini-columns, inorder to generate a profile of 384 different protein antigens.Antibodies are covalently coupled to agarose beads using standardwater-soluble carbodiimide chemistry. (March et al. (1974) A simplifiedmethod for cyanogen bromide activation of agarose for affinitychromatography. Anal Biochem. 60:149-152). The capacity of a small (75microliter) affinity column with one antibody is equal to approximately2×10¹¹ molecules of each protein. If multiplexing of reporter signals is256×, the maximum protein binding capacity will be approximately 10⁸molecules. The dynamic range of the assay will thus be 10,000 to100,000,000 molecules of protein.

Each of the protein preparations is tagged with a unique DNAoligonucleotides (coding tags), wherein a set of 64 different codingtags has the property of not being able to hybridize with each other.The protein preparation is reacted with 2-iminothiolane (Alagon andKing, (1980) Activation of polysaccharides with 2-iminothiolane and itsuses. Biochemistry. 19:4341-4345) to introduce reactive sulfhydrylgroups, if none is present. A DNA oligonucleotide (the coding tag),containing a reactive amino group at one of its termini is reacted witha heterobifunctional cross-linking reagent, such as SULFO-SMCC (Pierce,Inc.). The thiol-containing proteins are incubated together with theactivated oligonucleotide, to form a covalent protein-DNA adduct (thuslabeling the protein with a coding tag). For most protein molecules, theformation of this covalent adduct will not interfere with the capacityof the protein to associate with its cognate antibody. A total of 64protein preparations, each harboring covalently coupled unique codingtag sequences, are pooled together before being used for the multiplexedassay.

This example also involves the use of reporter molecules composed ofpeptide nucleic acid decoding tags and reporter signal peptides. Thedecoding tags comprise 64 different PNA sequences designed to beincapable of hybridizing to each other and additionally designed to becomplementary to each of the 64 aforementioned coding tags used forprotein labeling. The length of the PNA portion of the reporter moleculeis preferably 9 to 15 bases, and more preferably 10 to 11 bases. Thereporter molecules of this example also comprises 64 different sequencesof amino acids (the reporter signal peptides) which have the commonproperty of having the same mass, but being cleavable in such a way thatthey can be separated from each other after collision-inducedfragmentation.

1. Label each of 64 cell preps with a unique, non-self hybridizing, DNAoligonucleotide (coding tag), using SULFO-SMCC chemistry as indicatedabove.

2. Associate with affinity column on microtip containing specificantibody.

3. Repeat for 384 antibodies. That is, associate all of the labeled cellpreps to each of the 384 columns.

4. Pass all 64 reporter molecules through columns, to achievehybridization of PNA to DNA coding tags on proteins.

5. Elute proteins from column using acid matrix.

6. Separate and quantify reporter signals by MALDI-tandem massspectrometry where the amount of each different reporter signal ismeasured.

This example can be performed using peptide nucleic acid reportersignals (that is, reporter signals composed of peptide nucleic acid) toassociate directly with the coding tags. The reporter signals wouldcomprise 64 different PNA sequences of the same mass, designed to beincapable of hybridizing to each other, and additionally designed to becomplementary to each of the 64 aforementioned coding tags used forprotein labeling. The reporter signals could be easily dissociated fromthe coding tags (for detection) since they are only non-covalentlyassociated with the coding tags.

DD. Illustration 30: Multiple Samples Labeled with Different ReporterSignals

This illustration is an example of multiple sample labeling usingreporter signals where each sample is labeled with a different reportersignal. The samples are labeled via a DNA coding tag intermediate andthe samples are analyzed using an antibody array.

This illustration involves the use of an antibody microarray of 3200elements, constructed on a solid surface, the surface being compatiblewith analysis by mass spectrometry.

Each of the protein preparations is tagged with a unique DNAoligonucleotides (coding tags), wherein a set of 16 differentoligonucleotides has the property of not being able to hybridize witheach other. The protein preparation is reacted with 2-iminothiolane(Alagon and King, (1980) Activation of polysaccharides with2-iminothiolane and its uses. Biochemistry. 19:4341-4345) to introducereactive sulfhydryl groups, if none is present. A DNA oligonucleotide(coding tag), containing a reactive amino group at one of its termini isreacted with a heterobifunctional cross-linking reagent, such asSULFO-SMCC (Pierce, Inc.). The thiol-containing proteins are incubatedtogether with the activated oligonucleotide, to form a covalentprotein-DNA adduct, thus labeling the proteins with the coding tags. Formost protein molecules, the formation of this covalent adduct will notinterfere with the capacity of the protein to associate with its cognateantibody. A total of 16 protein preparations, each harboring covalentlycoupled unique DNA coding tag sequences, are pooled together beforebeing used for the multiplexed assay.

As in Illustration 29, this example also involves the use of PNA-peptidereporter signals reporter molecules. The PNA portions are decoding tagsand comprises 16 different PNA sequences, designed to be incapable ofhybridizing to each other, and additionally designed to be complementaryto sequences in each of the 16 aforementioned DNA tags used for proteinlabeling. The reporter signal portion of the reporter moleculescomprises 16 different sequences of amino acids which have the commonproperty of having the same mass, but being cleavable in such a way thatthey can be separated from each other after collision-inducedfragmentation.

An additional property of the 16 coding tags used for tagging each ofthe 16 protein samples is that each coding tag is able to associate witheight molecules of the reporter molecule. Each tagged protein in thesample will contain, on the average, one to three DNA coding tags. Thus,each protein will be able to associate with many (8 to 24) reportermolecules. This design results in increased signal intensity of reportersignals in the mass spectrometer.

1. Label each of 16 cell preps with a unique, non-self hybridizing, DNAoligonucleotide (coding tag), using SULFO-SMCC chemistry as indicatedabove.

2. Place the sample on a microarray containing 3200 immobilizedantibodies, the microarray being constructed on the surface of a platesuitable for reading on a mass spectrometer. Incubate for 2 hours at 37°C. Wash the surface to remove un-associated sample.

3. Contact the antibody microarray with a mixture of 16 PNA-peptidereporter signal reporter molecules. Wash to remove excess reportermolecules.

4. Coat the surface with matrix, and load the microarray into a MALDItandem mass spectrometer.

5. Separate and quantify reporter signals by MALDI-tandem massspectrometry where the amount of each different reporter signal ismeasured.

This example can be performed using peptide nucleic acid reportersignals (that is, reporter signals composed of peptide nucleic acid) toassociate directly with the coding tags. The reporter signals wouldcomprise 16 different PNA sequences of the same mass, designed to beincapable of hybridizing to each other, and additionally designed to becomplementary to each of the 16 aforementioned coding tags used forprotein labeling. The reporter signals could be easily dissociated fromthe coding tags (for detection) since they are only non-covalentlyassociated with the coding tags.

EXAMPLE

An important property of some of the disclosed reporter signals is theiruse in sets where the reporter signals all have a common property(allowing the reporter signals to be separated from the “junk” basedupon this common property) and where the reporter signals can besubsequently altered to allow the detection of the individual members ofthe set of reporter signals. A number of peptides were synthesized withparticular sequences and compositions in order to demonstrate themanipulation and analysis of reporter signals utilizing a tandem massspectrometer. For this example, a set of reporter signals of common massbut differing sequence was used. The reporter signals were fragmented toreveal a part of the sequence, and the reporter signal fragments weredetected. Use of reporter signals having a scissile, —DP—, bond wasdemonstrated.

The quantification of multiple proteins from a complex mixture has notbeen adequately performed in the field of proteomics. Singly chargedpeptides containing a C-terminal arginine in an ion trap willpreferentially fragment at the C-termini of aspartic acid or glutamicacid residues, and proline containing peptides will fragment at theN-termini of the proline residues (Qin and Chait, Collision-induceddissociation of singly charged peptide ions in a matrix-assisted laserdesorption ionization ion trap mass spectrometer. Int. J. Mass Spectrom.(Netherlands), 190-191:313-20 (1999)). These principles were used indesigning an exemplary set of peptide reporter signals making use of aDP amino acid sequence to test the collisional fragmentation at thescissile bond between the aspartic acid and proline.

A Micromass Q-TOF instrument (Micromass Inc., Beverly, Mass.) was usedin this example. Peptides for this example were synthesized by Fmocamino acid synthesis on a Rainin Symphony. The reaction scale was 25μmol. Crude synthesis products were used in this example. The disclosedmethod should tolerate dirty samples and complex mixtures.

A. Initial Peptides

Eight peptides that varied in the amino acid sequence and/orincorporated isotopes of specific amino acids were synthesized. Thesewere the reporter signals.

Modified amino acids were used to demonstrate differential distributionof mass by differential distribution of heavy isotope in reportersignals (heavy isotope mode), and reporter signals of differing sequencewere used to demonstrate differential distribution of mass bydifferential distribution of individual amino acids in reporter signals(variable sequence mode). Modified protected amino acids containingheavy stable isotopes were obtained from Cambridge Isotopes. The twoamino acids used here were 3-¹³C-Ala and 2-¹³C-Gly which are each oneDalton heavier than their natural amino acids. Fmoc protectedphosphorylated serine was used to further demonstrate the heavy isotopemode and also demonstrate the use of side chain modified amino acids.These peptides were synthesized with free NH₂ and free COOH on N and Ctermini, respectively, as shown in Table 4. TABLE 4 Expected Mode (H:Heavy primary Isotope, S: Peptide charged Scissile Bond, ID Peptidefragment C: Control) LAT3838 AGSLDPAGSLR PAGSLR⁺ C LAT3839A*G*S*LDPAGSLR PAGSLR⁺ H LAT3840 A*G*SLDPAGS*LR PAGS*LR⁺ H LAT3841A*GSLDPAG*S*LR PAG*S*LR⁺ H LAT3842 AGSLDPA*G*S*LR PA*G*S*LR⁺ H LAT3843AGSLADPGSLR PGSLR⁺ S LAT3844 AGSDPLAGSLR PLAGSLR⁺ S LAT3845 ADPGSLAGSLRPGSLAGSLR⁺ S LAT3846 AGSLAGSLDPR PR⁺ S

The peptides and primary charge fragments are SEQ ID NO:2 and aminoacids 6-11 of SEQ BD NO:2 for LAT3838, LAT3839, LAT3840, LAT3841, andLAT3842; SEQ ID NO:4 and amino acids 7-11 of SEQ ID NO:4 for LAT3843;SEQ ID NO:7 and amino acids 5-11 of SEQ ID NO:7 for LAT3844; SEQ ID NO:8and amino acids 3-11 of SEQ ID NO:8 for LAT3845; and SEQ BD NO:27 andamino acids 10-11 of SEQ ID NO:27 for LAT3846. In Table 4, an asteriskindicates cold heavy isotope amino acid in the case of gly and ala,phosphoserine in the case of serine. For LAT3839, the fragment isdistinguishable from control, but the parent ion is distinguishable.LAT3845 and LAT3846 exhibit an end of peptide effect.

B. Second Peptides

Based upon the results with the first peptides, six additional peptides(to serve as reporter signals) were synthesized to demonstrate furtherpoints, including reversing the sequence, adding a terminal cysteine (tofacilitate sulfur bridge covalent coupling) and addition of tyrosine (toallow for UV quantitation). These reporter signals are shown in Table 5.The set KER4086-KER4090 contain tryptophan to allow for quantitationusing UV absorbance. The peptides and primary charged fragments are SEQID NO:28 and amino acids 8-14 of SEQ ID NO:28 for KER4086; SEQ ID NO:29and amino acids 9-14 of SEQ ID NO:29 for KER4076; SEQ ID NO:30 and aminoacids 10-14 of SEQ ID NO:30 for KER4088; SEQ ID NO:31 and amino acids11-14 of SEQ ID NO:31 for KER4089; SEQ ID NO:32 and amino acids 12-14 ofSEQ ID NO:32 for KER4090; and SEQ ID NO:33 and amino acids 1-6 of SEQ IDNO:33 for KER4120. TABLE 5 Expected primary Peptide charged ID Peptidefragment Addresses KER4086 CGWAGSDPLAGSLR PLAGSLR⁺ UV quantitationKER4087 CGWAGSLDPAGSLR PAGSLR⁺ UV quantitation KER4088 CGWAGSLADPGSLRPGSLR⁺ UV quantitation KER4089 CGWAGSLAGDPSLR PSLR⁺ UV quantitationKER4090 CGWAGSLAGSDPLR PLR⁺ UV quantitation KER4120 RLSGADPLSGAWGCRLSGAD⁺ Sequence directionC. Instrumentation

The preferred mode for the disclosed method makes use of massspectrometry. Mass spectrometers consist of three major categories ofmodules: source, filter/ion guide/analyzer, and detector. Commonly usedsources for biological applications include Matrix Assisted LaserDesorption Ionization, MALDI, and Electrospray Ionization, ESI.Detectors on current instrumentation are generally Microchannel Plate,MCP, with a number of other detectors available. Between the source andthe detector are any number of filters, ion guides, collision cells,laser excitation regions, mass analyzers, etc.

The class of instrument used in this example is called tandem massspectrometer. The specific instrument used for these experiments isshown schematically in FIG. 1. A preferred spectrometer would have aMALDI source rather than the ESI source (MALDI tends to product singlycharged ions, ESI tens to produce multiply charged ions). The ESI sourceof the spectrometer used here served to provide a more stringentdemonstration of the disclosed method.

D. Results

1. DP Sequence, One Component

The first analysis was conducted using a single peptide (LAT3838) todemonstrate scissile bond cleavage in this instrument. To demonstratescissile bond cleavage, an approximate 1 mg/ml solution of a singlepeptide (LAT3838—dissolved in 50% acetonitrile/50% water/0.2% formicacid) was loaded into the mass spectrometer.

The sequence is AGSLDPAGSLR (SEQ ID NO:2) and was expected to fragmentto a single daughter PAGSLR⁺ (amino acids 6-11 of SEQ ID NO:2). Thecomplex ESI-TOF spectrum of AGSLDPAGSLR (SEQ ID NO:2) is shown in FIG.3. This spectrum was generated when all peptides (the complete peptideand any fragments) from the ESI source are passed through the firstresolving quadrupole, the collision cell, and into the TOF. The daughterESI-MS/MS spectrum is shown in FIG. 4. To produce this spectrum, theresolving quadrupole operated as a mass filter to select for the parentpeptide, allowing it to enter the collision cell while other peptideswere not able to pass into the collision cell. Once in the collisioncell, fragmentation occurred at the scissile DP bond as expected.

2. DP Sequence, Multiplexed

The set of peptides LAT3838 and LAT3843-3846 comprise an isobaric set ofreporter signals. That is, all of the reporter signals in this set havethe same mass-to-charge ratio. This set was mixed together inapproximately equal concentration. 7 mg of each peptide was dissolved in0.7 ml of 50% acetonitrile/50% water/0.2% formic acid. Dilutions of thestock samples were prepared and a final solution containing 0.1 μg/ml ofeach peptide was loaded into the mass spectrometer and subjected toESI-MS/MS analysis. The parent spectrum was comparable to that shown inFIG. 3, the daughter ion spectrum, shown in FIG. 5, exhibits all fivereporter signals at approximately equal amounts.

3. Heavy Isotopes

i. Phosphate Loss from Phosphorylated Serine

Spectra clearly show that phosphate loss is common in this system. Thisprovides increased sampling, with two data points generated per reportersignal. A typical example of loss of phosphate from the phosphorylatedserine is shown in FIG. 6.

ii. Stable Isotope Amino Acids

The stable isotopes incorporated into the reporter signals differed fromthe nature forms of the amino acids by 1 Dalton. As a consequence,naturally occurring heavy isotope peaks and the peak from the engineeredreporter signals are at the same (degenerate) mass-to-charge. A range ofmass-to-charge ratios is preferred for the disclosed method. Theresulting traces were analyzed in a straightforward manner (see Table6). It is clear from Table 6 and FIG. 7 that the “complicated peaks”correspond to three species and there are five measurements—thesimultaneous equations can be solved for each fragment. Additionally,the peaks near m/z=582 and the peaks near m/z=680 correspond to thefragment less phosphate and fragment respectively. This information isredundant (assuming the loss of phosphate is a random chance event) andmay be used to increase the quantitation confidence. TABLE 6 Observedm/z Species responsible for signal 600.34 LAT3839_(M) 601.33LAT3839_(M+1) 602.34 LAT3839_(M+2) 680.30 LAT3840_(M) 681.32LAT3840_(M+1) + LAT3841_(M) 682.30 LAT3840_(M+2) + LAT3841_(M+1) +LAT3842_(M) 683.31 LAT3841_(M+2) + LAT3842_(M+1) 684.31 LAT3842_(M+2)582.33 LAT3840_(M) − H₃PO₄ 583.33 LAT3840_(M+1) + LAT3841_(M) − H₃PO₄584.35 LAT3840_(M+2) + LAT3841_(M+1) + LAT3842_(M) − H₃PO₄ 585.35LAT3841_(M+2) + LAT3842_(M+1) − H₃PO₄ 568.35 LAT3842_(M+2) − H₃PO₄

In Table 6, the observed m/z corresponds to that in FIG. 7. Nomenclature(not industry standard): PeptideID_(M) is fragment which contains nonaturally occurring heavy isotope, PeptideID_(M+1) is the peak due tonaturally occurring single mass occurrences of one unit heavy isotope ofthe fragment, PeptideID_(M+2) is the peak due to naturally occurringdouble mass unit heavy isotopes plus naturally occurring instances oftwo single mass heavy isotopes. Effect only up to M+2 are consideredhere but the method is extensible.

4. Second Peptides

The second set of peptides (reporter signals) is shown in Table 5 andspecific aspects are addressed in these following sections. The dataindicate the tryptophan, the cysteine, and the additional glycine in thesecond set of peptides behave well in the disclosed method.

i. Effect of Sequence

Sulfur bridge covalent bonding of reporter signals to specific bindingmolecules can be used. To facilitate this chemistry it is advantageousto have a cysteine as a terminal residue. Additionally, because peptidesynthesis is from N to C terminus, if the cysteine is last on it can actas a purification element when the peptide is covalently attached to aspecific binding molecule (compare to amine linker last on duringoligonucleotide synthesis acts to purify the immobilizedoligonucleotide).

To demonstrate the effect a C-terminal cysteine, peptide KER4120(RLSGADPLSGAWGC; SEQ ID NO:33)) was synthesized which is preciselypeptide KER4087 (CGWAGSLDPAGSLR; SEQ ID NO:29) in reverse sequence. Thefragmentation pattern of KER4120 was similar to that of KER4087. This isconsistent with a conclusion that synthesis of the peptide with thearginine on the N-terminus results in a peptide with a similarfragmentation pattern.

5. Complex Mixture

Clear demonstration of the power of the disclosed method is seen in anexample measurement in a complex mixture. To generate a reasonablycomplex mixture Bovine Serum Albumin (BSA) (66 kDa) and creatinephosphokinase (84 kDa) were digested using trypsin. Five peptides(KER4086 to KER4090) were added to the digestion mixture (to a finalconcentration of 0.1 μg/ml of each peptide). The resulting mass spectrumfor this complex mixture is shown in FIG. 8. As can be seen, the mixtureis quite complex, showing peaks at a wide variety of mass-to-chareratios. The spectrum following selection of the common mass-to-chargeratio is shown in FIG. 9. As can be seen, the filtering (selection) stepproduces a dramatic (essentially complete) reduction in complexity. Thespectrum following fragmentation of the selected mass-to-charge fractionis shown in FIG. 10. As can be seen, clear peaks, all at nearly the samelevel (as expected based on equal amounts of starting material for eachreporter signal), appear for each of the five expected reporter signalfragments (each having a distinctive mass-to-charge ratio. FIGS. 8through 10 give a powerful representation of the progression from acomplex mixture to the identification and determination of relativeconcentrations of specific labels.

It is understood that the disclosed invention is not limited to theparticular methodology, protocols, and reagents described as these mayvary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention which will belimited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a ”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “ahost cell” includes a plurality of such host cells, reference to “theantibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Disclosed are the components to be used to prepare the disclosedcompositions as well as the compositions themselves and to be usedwithin the methods disclosed herein. These and other materials aredisclosed herein, and it is understood that when combinations, subsets,interactions, groups, etc. of these materials are disclosed that whilespecific reference of each various individual and collective permutationof these compounds may not be explicitly disclosed, each is specificallycontemplated and described herein. For example, if a particular type ofreporter signal is disclosed and discussed in the context of some modesand embodiments of the disclosed method, specifically contemplated iseach and every combination and permutation of the reporter signal andthe various forms and embodiments of the disclosed method that arepossible unless specifically indicated to the contrary. Thus, if a classof molecules A, B, and C are disclosed as well as a class of moleculesD, E, and F and an example of a combination molecule, A-D is disclosed,then even if it each is not individually recited each is individuallyand collectively contemplated meaning combinations, A-E, A-F, B-D, B-E,B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset orcombination of these is also disclosed. Thus, for example, the sub-groupof A-E, B-F, and C-E would be considered disclosed. This concept appliesto all aspects of this application including, but not limited to, stepsin methods of making and using the disclosed compositions. Thus, ifthere are a variety of additional steps that can be performed it isunderstood that each of these additional steps can be performed with anyspecific embodiment or combination of embodiments of the disclosedmethods.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are as described. Publications cited herein andthe material for which they are cited are specifically incorporated byreference. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A set of reporter signals comprising a plurality of reporter signals,wherein the reporter signals have a common property, wherein the commonproperty allows the reporter signals to be distinguished or separatedfrom molecules lacking the common property, wherein the reporter signalscan be altered, wherein the altered forms of each reporter signal can bedistinguished from every other altered form of reporter signal.
 2. Theset of claim 1 wherein the common property is mass-to-charge ratio,wherein the reporter signals are altered by altering their mass, whereinthe altered forms of the reporter signals can be distinguished viadifferences in the mass-to-charge ratio of the altered forms of reportersignals.
 3. The set of claim 2 wherein the mass of the reporter signalsis altered by fragmentation.
 4. The set of claim 2 wherein alteration ofthe reporter signals also alters their charge.
 5. The set of claim 1wherein the common property is mass-to-charge ratio, wherein thereporter signals are altered by altering their charge, wherein thealtered forms of the labeled proteins can be distinguished viadifferences in the mass-to-charge ratio of the altered forms of reportersignals.
 6. The set of claim 1 wherein the set comprises two or more,three or more, four or more, five or more, six or more, seven or more,eight or more, nine or more, ten or more, twenty or more, thirty ormore, forty or more, fifty or more, sixty or more, seventy or more,eighty or more, ninety or more, or one hundred or more differentreporter signals.
 7. The set of claim 6 wherein the set comprises ten ormore different reporter signals.
 8. The set of claim 1 wherein thereporter signals are peptides, oligonucleotides, carbohydrates,polymers, oligopeptides, or peptide nucleic acids.
 9. The set of claim 1wherein the reporter signals are associated with, or coupled to,specific binding molecules, wherein each reporter signal is associatedwith, or coupled to, a different specific binding molecule.
 10. The setof claim 1 wherein the reporter signals are associated with, or coupledto, decoding tags, wherein each reporter signal is associated with, orcoupled to, a different decoding tag.
 11. The set of claim 1 wherein thereporter signals comprise peptides, wherein the peptides have the samemass-to-charge ratio.
 12. The set of claim 11 wherein the peptides havethe same amino acid composition.
 13. The set of claim 12 wherein thepeptides have the same amino acid sequence.
 14. The set of claim 13wherein each peptide contains a different distribution of heavyisotopes.
 15. The set of claim 13 wherein each reporter signal peptidecontains a different distribution of substituent groups.
 16. The set ofclaim 12 wherein each peptide has a different amino acid sequence. 17.The set of claim 12 wherein each peptide has a labile or scissile bondin a different location.
 18. The set of claim 1 wherein the reportersignals are coupled to the proteins or peptides.
 19. The set of claim 1wherein the common property allows the labeled proteins to bedistinguished or separated from molecules lacking the common property.20. The set of claim 1 wherein the common property is not an affinitytag.
 21. The set of claim 20 wherein one or more affinity tags areassociated with the reporter signals.
 22. A method comprising (a)separating a set of reporter signals, where each reporter signal has acommon property, from molecules lacking the common property, (b)altering the reporter signals, (c) detecting and distinguishing thealtered forms the reporter signals from each other.
 23. The method ofclaim 22 wherein the common property is mass-to-charge ratio, whereinthe reporter signals are altered by altering their mass, wherein thealtered forms of the reporter signals are distinguished via differencesin the mass-to-charge ratio of the altered forms of reporter signals.24. The method of claim 23 wherein the mass of the reporter signals isaltered by fragmentation.
 25. The method of claim 23 wherein thereporter signals are altered by cleavage at a photocleavable amino acid.26. The method of claim 23 wherein the reporter signals are fragmentedin a collision cell.
 27. The method of claim 23 wherein the reportersignals are fragmented at an asparagine-proline bond.
 28. The method ofclaim 23 wherein alteration of the reporter signals also alters theircharge.
 29. The method of claim 22 wherein the common property ismass-to-charge ratio, wherein the reporter signals are altered byaltering their charge, wherein the altered forms of the labeled proteinscan be distinguished via differences in the mass-to-charge ratio of thealtered forms of reporter signals.
 30. The method of claim 22 whereinthe set of reporter signals comprises two or more, three or more, fouror more, five or more, six or more, seven or more, eight or more, nineor more, ten or more, twenty or more, thirty or more, forty or more,fifty or more, sixty or more, seventy or more, eighty or more, ninety ormore, or one hundred or more different reporter signals.
 31. The methodof claim 30 wherein the set of reporter signals comprises ten or moredifferent reporter signals.
 32. The method of claim 22 wherein thereporter signals are peptides, oligonucleotides, carbohydrates,polymers, oligopeptides, or peptide nucleic acids.
 33. The method ofclaim 22 wherein the reporter signals are associated with, or coupledto, specific binding molecules, wherein each reporter signal isassociated with, or coupled to, a different specific binding molecule.34. The method of claim 22 wherein the reporter signals are associatedwith, or coupled to, decoding tags, wherein each reporter signal isassociated with, or coupled to, a different decoding tag.
 35. The methodof claim 22 wherein the reporter signals comprise peptides, wherein thepeptides have the same mass-to-charge ratio.
 36. The method of claim 35wherein the peptides have the same amino acid composition.
 37. Themethod of claim 36 wherein the peptides have the same amino acidsequence.
 38. The method of claim 37 wherein each peptide contains adifferent distribution of heavy isotopes.
 39. The method of claim 37wherein each reporter signal peptide contains a different distributionof substituent groups.
 40. The method of claim 36 wherein each peptidehas a different amino acid sequence.
 41. The method of claim 36 whereineach peptide has a labile or scissile bond in a different location. 42.The method of claim 22 wherein the reporter signals are coupled to theproteins or peptides.
 43. The method of claim 22 wherein the commonproperty allows the labeled proteins to be distinguished or separatedfrom molecules lacking the common property.
 44. The method of claim 22wherein the common property is not an affinity tag.
 45. The method ofclaim 44 wherein one or more affinity tags are associated with thereporter signals.
 46. The method of claim 22 further comprising, priorto step (a), associating the reporter signals with one or more analytes,wherein each reporter signal is associated with, or coupled to, adifferent specific binding molecule, wherein each specific bindingmolecule can interact specifically with a different one of the analytes,wherein the reporter signals are associated with the analytes viainteraction of the specific binding molecules with the analytes.
 47. Themethod of claim 22 further comprising, prior to step (a), associatingone or more reporter signals with one or more proteins, one or morepeptides, or one or more proteins and peptides from each of one or moresamples.
 48. The method of claim 22 wherein the reporter signals areassociated with a single sample.
 49. The method of claim 48 wherein thesample is produced by a separation procedure, wherein the separationprocedure comprises liquid chromatography, gel electrophoresis,two-dimensional chromatography, two-dimensional gel electrophoresis,isoelectric focusing, thin layer chromatography, centrifugation,filtration, ion chromatography, immunoaffinity chromatography, membraneseparation, or a combination of these.
 50. The method of claim 22wherein steps (a) through (c) are repeated one or more times using adifferent set of reporter signals each time.
 51. The method of claim 50wherein, prior to step (a), the different sets of reporter signals areassociated with different samples.
 52. The method of claim 51 whereinthe different sets of reporter signals each comprise the same reportersignals.
 53. The method of claim 51 wherein the samples are produced bya separation procedure, wherein the separation procedure comprisesliquid chromatography, gel electrophoresis, two-dimensionalchromatography, two-dimensional gel electrophoresis, isoelectricfocusing, thin layer chromatography, centrifugation, filtration, ionchromatography, immunoaffinity chromatography, membrane separation, or acombination of these.
 54. The method of claim 51 wherein the differentsamples are from the same protein sample.
 55. The method of claim 54wherein the different samples are obtained at different times.
 56. Themethod of claim 51 wherein the different samples are from the same typeof organism.
 57. The method of claim 51 wherein the different samplesare from the same type of tissue.
 58. The method of claim 51 wherein thedifferent samples are from the same organism.
 59. The method of claim 58wherein the different samples are obtained at different times.
 60. Themethod of claim 51 wherein the different samples are from differentorganisms.
 61. The method of claim 51 wherein the different samples arefrom different types of tissues.
 62. The method of claim 51 wherein thedifferent samples are from different species of organisms.
 63. Themethod of claim 51 wherein the different samples are from differentstrains of organisms.
 64. The method of claim 51 wherein the differentsamples are from different cellular compartments.
 65. The method ofclaim 51 further comprising identifying or preparing proteins orpeptides corresponding the proteins or peptides present in one samplebut not present in another sample.
 66. The method of claim 51 furthercomprising determining the relative amount of proteins or peptides inthe different samples.
 67. The method of claim 50 wherein the sets ofreporter signals each contain a single reporter signal.
 68. The methodof claim 22 wherein not all of the reporter signals in the set aredistinguished or separated from molecules lacking the common property,not all of the reporter signals are altered, and not all of the alteredforms of the reporter signals are detected at the same time.
 69. Themethod of claim 68 wherein all of the reporter signals in the set aredistinguished or separated from molecules lacking the common property,all of the reporter signals are altered, and all of the altered forms ofthe reporter signals are detected at different times.
 70. The method ofclaim 22 wherein steps (a) through (c) are performed separately for eachreporter signal.
 71. The method of claim 22 wherein the altered forms ofthe labeled proteins detected collectively constitutes a catalog ofproteins.
 72. The method of claim 22 wherein steps (b) and (c) areperformed simultaneously.
 73. The method of claim 22 wherein the alteredforms of the target protein fragments are detecting using massspectrometry.
 74. The method of claim 73 wherein the steps are performedwith a tandem mass spectrometer.
 75. The method of claim 74 wherein thetandem mass spectrometer comprises a first stage and a last stage,wherein step (a) is performed using the first stage of the tandem massspectrometer to select ions in a narrow mass-to-charge range, whereinstep (b) is performed by collision with a gas, and wherein step (c) isperformed using the final stage of the tandem mass spectrometer.
 76. Themethod of claim 75 where the first stage of the tandem mass spectrometeris a quadrupole mass filter.
 77. The method of claim 76 where the finalstage of the tandem mass spectrometer is a time of flight analyzer. 78.The method of claim 75 where the final stage of the tandem massspectrometer is a time of flight analyzer.
 79. The method of claim 74wherein the mass-to-charge range is varied to cover the mass-to-chargeratio of each of the target protein fragments.
 80. A kit comprising (a)a set of reporter molecules, wherein each reporter molecule comprises areporter signal and a decoding tag, wherein the reporter signals have acommon property, wherein the common property allows the reporter signalsto be distinguished or separated from molecules lacking the commonproperty, wherein the reporter signals can be altered, wherein thealtered forms of each reporter signal can be distinguished from everyother altered form of reporter signal, wherein each different reportermolecule comprises a different decoding tag and a different reportersignal, (b) a set of coding molecules, wherein each coding moleculecomprises a specific binding molecule and a coding tag, wherein eachspecific binding molecule can interact specifically with a differentanalyte, wherein each coding tag can interact specifically with adifferent decoding tag.
 81. A method comprising (a) separating one ormore reporter signals, where each reporter signal has a common property,from molecules lacking the common property in each of a plurality ofsamples, (b) altering the reporter signals, (c) detecting anddistinguishing the altered forms the reporter signals from each other.82. A set of labeled proteins wherein each labeled protein comprises aprotein or peptide and a reporter signal attached to the protein orpeptide, wherein the reporter signals have a common property, whereinthe common property allows the labeled proteins comprising the sameprotein or peptide to be distinguished or separated from moleculeslacking the common property, wherein the reporter signals can bealtered, wherein the altered forms of each reporter signal can bedistinguished from every other altered form of reporter signal, whereinalteration of the reporter signals alters the labeled proteins, whereinaltered forms of each labeled protein can be distinguished from everyother altered form of labeled protein.
 83. A kit comprising a set ofreporter molecules, wherein each reporter molecule comprises a reportersignal and a coupling tag, wherein the reporter signals have a commonproperty, wherein the common property allows the reporter signals to bedistinguished or separated from molecules lacking the common property,wherein the reporter signals can be altered, wherein the altered formsof each reporter signal can be distinguished from every other alteredform of reporter signal, wherein each different reporter moleculecomprises a different coupling tag and a different reporter signal. 84.A set of labeled proteins wherein each labeled protein comprises aprotein or peptide and a reporter signal attached to the protein orpeptide, wherein the labeled proteins have a common property, whereinthe common property allows the labeled proteins comprising the sameprotein or peptide to be distinguished or separated from moleculeslacking the common property, wherein the reporter signals can bealtered, wherein the altered forms of each reporter signal can bedistinguished from every other altered form of reporter signal, whereinalteration of the reporter signals alters the labeled proteins, whereinaltered forms of each labeled protein can be distinguished from everyother altered form of labeled protein.
 85. A set of labeled proteinswherein each labeled protein comprises a protein or peptide and areporter signal attached to the protein or peptide, wherein the reportersignals can be altered, wherein the altered forms of each reportersignal can be distinguished from every other altered form of reportersignal, wherein alteration of the reporter signals alters the labeledproteins, wherein altered forms of each labeled protein can bedistinguished from every other altered form of labeled protein.
 86. Aset of labeled proteins wherein each labeled protein comprises a proteinor peptide and a reporter signal attached to the protein or peptide,wherein the reporter signals have a common property, wherein the commonproperty allows the labeled proteins comprising the same protein orpeptide to be distinguished or separated from molecules lacking thecommon property.
 87. A labeled protein wherein the labeled proteincomprises a protein or peptide and a reporter signal attached to theprotein or peptide, wherein the labeled protein has a common property,wherein the common property allows the labeled protein to bedistinguished or separated from molecules lacking the common property,wherein the reporter signal can be altered, wherein alteration of thereporter signals alters the labeled protein, wherein altered form of thelabeled protein can be distinguished from the unaltered form of labeledprotein.
 88. A method comprising (a) separating a set of labeledproteins, wherein each labeled protein comprises a protein or peptideand a reporter signal attached to the protein or peptide, wherein eachreporter signal has a common property, wherein the common propertyallows the labeled proteins comprising the same protein or peptide to bedistinguished or separated from molecules lacking the common property,(b) altering the reporter signals, thereby altering the labeledproteins, (c) detecting and distinguishing the altered forms of thelabeled proteins from each other.
 89. The method of claim 88 furthercomprising, prior to step (a), attaching the reporter signals to one ormore proteins, one or more peptides, or one or more proteins andpeptides.
 90. The method of claim 89 wherein steps are repeated one ormore times using a different set of reporter signals each time.
 91. Themethod of claim 90 wherein, prior to step (a), the different sets ofreporter signals are attached to proteins or peptides in differentsamples.
 92. The method of claim 91 wherein the different sets ofreporter signals each comprise the same reporter signals.
 93. The methodof claim 90 wherein the sets of reporter signals each contain a singlereporter signal.
 94. A set of labeled proteins wherein each labeledprotein comprises a protein or a peptide and a reporter signal attachedto the protein or peptide, wherein the reporter signals comprisepeptides, wherein the reporter signal peptides have the samemass-to-charge ratio.
 95. A method comprising (a) separating one or morelabeled proteins, wherein each labeled protein comprises a protein orpeptide and a reporter signal attached to the protein or peptide,wherein each reporter signal has a common property, wherein the commonproperty allows the labeled proteins comprising the same protein orpeptide to be distinguished or separated from molecules lacking thecommon property in each of one or more samples, (b) altering thereporter signals, thereby altering the labeled proteins, (c) detectingand distinguishing the altered forms the labeled proteins from eachother.
 96. The method of claim 95 wherein the pattern of the presence,amount, presence and amount, or absence of labeled proteins in one ofthe samples constitutes a catalog of proteins in the sample.
 97. Themethod of claim 96 wherein the pattern of the presence, amount, presenceand amount, or absence of labeled proteins in a second one of thesamples constitutes a catalog of proteins in the second sample, whereinthe catalog of proteins in the first sample is a first catalog and thecatalog of proteins in the second sample is a second catalog, the methodfurther comprising comparing the first catalog and the second catalog.98. A method comprising (a) separating a set of labeled proteins,wherein each labeled protein comprises a protein or peptide and areporter signal attached to the protein or peptide, wherein each labeledprotein has a common property, wherein the common property allows thelabeled proteins comprising the same protein or peptide to bedistinguished or separated from molecules lacking the common property,(b) altering the reporter signals, thereby altering the labeledproteins, (c) detecting and distinguishing the altered forms of thelabeled proteins from each other.
 99. A method comprising (a) alteringlabeled proteins, wherein each labeled protein comprises a protein orpeptide and a reporter signal attached to the protein or peptide,wherein the labeled proteins are altered by altering the reportersignals, (b) detecting and distinguishing the altered forms of thelabeled proteins from each other.
 100. A method of detecting a proteinor peptide, the method comprising (a) altering a labeled protein,wherein the labeled protein comprises a protein or peptide and areporter signal attached to the protein or peptide, wherein the labeledprotein is altered by altering the reporter signal, (b) detecting anddistinguishing the altered form of the labeled protein from theunaltered form of labeled protein.
 101. The method of claim 100 furthercomprising, detecting the unaltered form of labeled protein.
 102. Amethod of detecting a protein, the method comprising, detecting alabeled protein, wherein the labeled protein comprises a protein orpeptide and a reporter signal attached to the protein or peptide,wherein the labeled protein is altered by altering the reporter signal,detecting an altered form of the labeled protein, wherein the labeledprotein is altered by altering the reporter signal, and identifying theprotein based on the characteristics of the labeled protein and alteredform of the labeled protein.
 103. The method of claim 102 wherein thelabeled protein and altered form of the labeled protein are detected bydetecting the mass-to-charge ratio of the labeled protein and themass-to-charge ratio of the altered form of the labeled protein or themass-to-charge ratio of the altered form of the reporter signal.
 104. Amethod comprising (a) separating one or more labeled proteins from othermolecules, wherein the labeled proteins are derived from one or moresamples, wherein each labeled protein comprises a protein or peptide anda reporter signal attached to the protein or peptide, (b) altering thereporter signals, thereby altering the labeled proteins, (c) detectingand distinguishing the altered forms the labeled proteins from eachother.
 105. The method of claim 104 further comprising, prior to step(a), associating one or more reporter signals with one or more proteins,one or more peptides, or one or more proteins and peptides from each ofthe one or more samples.
 106. The method of claim 104 wherein the one ormore labeled proteins are derived from a single sample.
 107. The methodof claim 106 wherein a single labeled protein is distinguished orseparated from other molecules.
 108. The method of claim 106 wherein aplurality of labeled proteins are distinguished or separated from othermolecules.
 109. The method of claim 106 wherein the detected alteredforms of the labeled proteins constitute a catalog of proteins in thesample.
 110. The method of claim 104 wherein one or more labeledproteins are derived from each of a plurality of samples.
 111. Themethod of claim 110 wherein a single labeled protein derived from eachof the samples is distinguished or separated from other molecules. 112.The method of claim 110 wherein a plurality of labeled proteins derivedfrom each of the samples are distinguished or separated from othermolecules.
 113. The method of claim 110 wherein the detected alteredforms of the labeled proteins derived from each sample constitute acatalog of proteins in the sample.
 114. A catalog of proteins andpeptides comprising, proteins and peptides in a sample detected by (a)separating one or more labeled proteins from other molecules, whereinthe labeled proteins are derived from the sample, wherein each labeledprotein comprises a protein or peptide and a reporter signal attached tothe protein or peptide, (b) altering the reporter signals, therebyaltering the labeled proteins, (c) detecting and distinguishing thealtered forms the labeled proteins from each other.
 115. A catalog ofproteins and peptides comprising, proteins and peptides in one or moresamples detected by (a) separating one or more labeled proteins fromother molecules, wherein the labeled proteins are derived from the oneor more samples, wherein each labeled protein comprises a protein orpeptide and a reporter signal attached to the protein or peptide, (b)altering the reporter signals, thereby altering the labeled proteins,(c) detecting and distinguishing the altered forms the labeled proteinsfrom each other.
 116. A method of producing a protein signature, themethod comprising (a) treating a protein sample to produce proteinfragments, wherein the protein fragments comprise a set of targetprotein fragments, wherein the target protein fragments can be altered,wherein the altered forms of the target protein fragments can bedistinguished from the other altered forms of the target proteinfragments, (b) mixing the target protein fragments with a set ofreporter signal calibrators, wherein each target protein fragment sharesa common property with at least one of the reporter signal calibrators,wherein the common property allows the target protein fragments andreporter signal calibrators having the common property to bedistinguished or separated from molecules lacking the common property,wherein the target protein fragment and reporter signal calibrator thatshare a common property correspond to each other, wherein the reportersignal calibrators can be altered, wherein the altered form of eachreporter signal calibrator can be distinguished from the altered form ofthe target protein fragment with which the reporter signal calibratorshares a common property, (c) separating the target protein fragmentsand reporter signal calibrators from other molecules based on the commonproperties of the target protein fragments and reporter signalcalibrators, (d) altering the target protein fragments and reportersignal calibrators, (e) detecting the altered forms of the targetprotein fragments and reporter signal calibrators, wherein the presence,absence, amount, or presence and amount of the altered forms of thetarget protein fragments indicates the presence, absence, amount, orpresence and amount in the protein sample of the target proteinfragments from which the altered forms of the target protein fragmentsare derived, wherein the presence, absence, amount, or presence andamount of the target protein fragments in the protein sample constitutesa protein signature of the protein sample.
 117. The method of claim 116wherein a predetermined amount of each reporter signal calibrator ismixed with the target protein fragments, wherein the amount of eachaltered form of reporter signal calibrator detected provides a standardfor assessing the amount of the altered form of the corresponding targetprotein fragment.
 118. The method of claim 117 wherein the amount of atleast two of the reporter signal calibrators is different.
 119. Themethod of claim 117 wherein the relative amount each reporter signalcalibrator is based on the relative amount of each corresponding targetprotein fragment expected to be in the protein sample.
 120. The methodof claim 117 wherein the amount of each of the reporter signalcalibrators is the same.
 121. The method of claim 116 wherein theprotein fragments are produced by protease digestion of the proteinsample.
 122. The method of claim 121 wherein the protein fragments areproduced by digestion of the protein sample with a serine protease. 123.The method of claim 122 wherein the serine protease is trypsin.
 124. Themethod of claim 121 wherein the protein fragments are produced bydigestion of the protein sample with Factor Xa or Enterokinase.
 125. Themethod of claim 116 wherein the protein fragments are produced bycleavage at a photocleavable amino acid.
 126. The method of claim 116wherein the set of target protein fragments comprises two or more, threeor more, four or more, five or more, six or more, seven or more, eightor more, nine or more, ten or more, twenty or more, thirty or more,forty or more, fifty or more, sixty or more, seventy or more, eighty ormore, ninety or more, or one hundred or more different target proteinfragments.
 127. The method of claim 116 further comprising comparing theprotein signature to one or more other protein signatures.
 128. Themethod of claim 116 wherein at least one of the target protein fragmentscomprises at least one modified amino acid.
 129. The method of claim 128wherein the modified amino acid is a phosphorylated amino acid, anacylated amino acid, or a glycosylated amino acid.
 130. The method ofclaim 128 wherein at least one of the target protein fragments is thesame as the target protein fragment comprising the modified amino acidexcept for the modified amino acid.
 131. The method of claim 116 furthercomprising performing steps (a) through (e) on a control protein sample,identifying differences between the protein signatures produced from theprotein sample and the control protein sample.
 132. The method of claim116 further comprising performing steps (a) through (e) on a pluralityof protein samples.
 133. The method of claim 132 further comprisingidentifying differences between the protein signatures produced from theprotein samples.
 134. The method of claim 132 further comprisingperforming steps (a) through (e) on a control protein sample,identifying differences between the protein signatures produced from theprotein samples and the control protein sample.
 135. The method of claim134 wherein the differences are differences in the presence, amount,presence and amount, or absence of target protein fragments in theprotein samples and the control protein sample.
 136. The method of claim132 wherein the steps (a) through (e) are performed on a control proteinsample and a tester protein sample, wherein the tester protein sample,or the source of the tester protein sample, is treated, prior to step(a), so as to destroy, disrupt or eliminate one or more proteinmolecules in the tester protein sample, wherein the target proteinfragments corresponding to the destroyed, disrupted, or eliminatedprotein molecules will be produced from the control protein sample butnot the tester protein sample.
 137. The method of claim 136 wherein thetester protein sample is treated so as to destroy, disrupt or eliminateone or more protein molecules in the tester protein sample.
 138. Themethod of claim 137 wherein one or more protein molecules in the testersample are eliminated by separating the one or more protein moleculesfrom the tester protein sample.
 139. The method of claim 138 wherein theone or more protein molecules are separated by affinity separation. 140.The method of claim 136 wherein the source of the tester protein sampleis treated so as to destroy, disrupt or eliminate one or more proteinmolecules in the tester protein sample.
 141. The method of claim 140wherein the treatment of the source is accomplished by exposing cellsfrom which the tester sample will be derived with a compound,composition, or condition that will reduce or eliminate expression ofone or more genes.
 142. The method of claim 136 further comprisingidentifying differences in the target protein fragments in the controlprotein sample and tester protein sample.
 143. The method of claim 132further comprising identifying differences between the target proteinfragments in the protein samples.
 144. The method of claim 132 whereinthe plurality of protein samples are produced by a separation procedure,wherein the separation procedure comprises liquid chromatography, gelelectrophoresis, two-dimensional chromatography, two-dimensional gelelectrophoresis, isoelectric focusing, thin layer chromatography,centrifugation, filtration, ion chromatography, immunoaffinitychromatography, membrane separation, or a combination of these.
 145. Themethod of claim 144 wherein the protein samples are different fractionsor samples produced by the same separation procedure.
 146. The method ofclaim 116 further comprising performing steps (a) through (e) on asecond protein sample.
 147. The method of claim 116 further comprisingproducing a second protein signature from a second protein sample andcomparing the first protein signature and second protein signature,wherein differences in the first and second protein signatures indicatedifferences in source or condition of the source of the first and secondprotein samples.
 148. The method of claim 116 further comprisingproducing a second protein signature from a second protein sample andcomparing the first protein signature and second protein signature,wherein differences in the first and second protein signatures indicatedifferences in protein modification of the first and second proteinsamples.
 149. The method of claim 148 wherein the second protein sampleis a sample from the same type of cells as the first protein sampleexcept that the cells from which the first protein sample is derived aremodification-deficient relative to the cells from which the secondprotein sample is derived.
 150. The method of claim 148 wherein thesecond protein sample is a sample from a different type of cells thanthe first protein sample, and wherein the cells from which the firstprotein sample is derived are modification-deficient relative to thecells from which the second protein sample is derived.
 151. The methodof claim 116 wherein the protein sample is derived from one or morecells.
 152. The method of claim 151 wherein the protein signatureindicates the physiological state of the cells.
 153. The method of claim151 wherein the protein signature indicates the effect of a treatment ofthe cells.
 154. The method of claim 153 wherein the cells are derivedfrom an organism, wherein the cells are treated by treating theorganism.
 155. The method of claim 154 wherein the organism is treatedby administering a compound to the organism.
 156. The method of claim154 wherein the organism is human.
 157. The method of claim 116 whereinthe protein sample is produced by a separation procedure, wherein theseparation procedure comprises liquid chromatography, gelelectrophoresis, two-dimensional chromatography, two-dimensional gelelectrophoresis, isoelectric focusing, thin layer chromatography,centrifugation, filtration, ion chromatography, immunoaffinitychromatography, membrane separation, or a combination of these.
 158. Themethod of claim 116 wherein the set of reporter signal calibratorsconsists of a single reporter signal calibrator.
 159. The method ofclaim 158 wherein the protein signature of the protein sample representsthe presence, absence, amount, or presence and amount of the targetprotein fragment in the protein sample that corresponds to the reportersignal calibrator.
 160. A method of producing a protein signature, themethod comprising detecting altered forms of target protein fragmentsand reporter signal calibrators, wherein the altered forms of the targetprotein fragments can be distinguished from the other altered forms ofthe target protein fragments, wherein each target protein fragmentshares a common property with at least one of the reporter signalcalibrators, wherein the common property allows the target proteinfragments and reporter signal calibrators having the common property tobe distinguished or separated from molecules lacking the commonproperty, wherein the target protein fragment and reporter signalcalibrator that share a common property correspond to each other,wherein the altered form of each reporter signal calibrator can bedistinguished from the altered form of the target protein fragment withwhich the reporter signal calibrator shares a common property, whereinthe presence, absence, amount, or presence and amount of the alteredforms of the target protein fragments indicates the presence, absence,amount, or presence and amount in a protein sample of the target proteinfragments from which the altered forms of the target protein fragmentsare derived, wherein the presence, absence, amount, or presence andamount of the target protein fragments in the protein sample constitutesa protein signature of the protein sample.
 161. The method of claim 160wherein the target protein fragments and reporter signal calibrators aredistinguished or separated from other molecules based on the commonproperties of the target protein fragments and reporter signalcalibrators.
 162. The method of claim 161 wherein the target proteinfragments and reporter signal calibrators are altered followingseparation.
 163. The method of claim 160 wherein the target proteinfragments are produced by treating the protein sample.
 164. A method ofproducing a protein signature, the method comprising (a) treating aprotein sample to produce protein fragments, wherein the proteinfragments comprise a set of target protein fragments, wherein the targetprotein fragments can be altered, wherein the altered forms of thetarget protein fragments can be distinguished from the other alteredforms of the target protein fragments, (b) separating the target proteinfragments from other protein fragments in the protein sample, (c)altering the target protein fragments, (d) detecting the altered formsof the target protein fragments, wherein the presence, absence, amount,or presence and amount of the altered forms of the target proteinfragments indicates the presence, absence, amount, or presence andamount in the protein sample of the target protein fragments from whichthe altered forms of the target protein fragments are derived, whereinthe presence, absence, amount, or presence and amount of the targetprotein fragments in the protein sample constitutes a protein signatureof the protein sample.
 165. The method of claim 164 further comprising,prior to or simultaneous with step (b), mixing the target proteinfragments with a set of reporter signal calibrators, wherein each targetprotein fragment shares a common property with at least one of thereporter signal calibrators, wherein the common property allows thetarget protein fragments and reporter signal calibrators having thecommon property to be distinguished or separated from molecules lackingthe common property, wherein the reporter signal calibrators can bealtered, wherein the altered form of each reporter signal calibrator canbe distinguished from the altered form of the target protein fragmentwith which the reporter signal calibrator shares a common property. 166.A method of producing a protein signature, the method comprising (a)separating a plurality of target protein fragments from other proteinfragments in a protein sample, (b) altering the target proteinfragments, (c) detecting the altered forms of the target proteinfragments, wherein the presence, absence, amount, or presence and amountof the altered forms of the target protein fragments indicates thepresence, absence, amount, or presence and amount in the protein sampleof the target protein fragments from which the altered forms of thetarget protein fragments are derived, wherein the presence, absence,amount, or presence and amount of the target protein fragments in theprotein sample constitutes a protein signature of the protein sample.167. A method of analyzing a protein sample, the method comprising (a)mixing a protein sample with a predetermined amount of a reporter signalcalibrator, wherein the protein sample has a known amount of protein,wherein the protein sample comprises a target protein fragment, whereinthe target protein fragment can be altered, wherein the reporter signalcalibrator can be altered, wherein the altered form of the reportersignal calibrator can be distinguished from the altered form of thetarget protein fragment, (b) altering the target protein fragment andreporter signal calibrator, (c) detecting the altered forms of thetarget protein fragment and reporter signal calibrator.
 168. The methodof claim 167 further comprising determining the ratio of the amount ofthe target protein fragment and the amount of the reporter signalcalibrator detected, and comparing the determined ratio with thepredicted ratio of the amount of the target protein fragment and theamount of the reporter signal calibrator, wherein the predicted ratio isbased on the predicted amount of target protein fragment in the proteinsample and the predetermined amount of reporter signal calibrator,wherein the predicted amount of target protein fragment is the amount oftarget protein fragment the protein sample would have if the knownamount of protein in the protein sample consisted of the target proteinfragment, wherein the difference between the determined ratio and thepredicted ratio is a measure of the purity of the protein sample for thetarget protein fragment, wherein the closer the determined ratio is tothe predicted ratio, the purer the protein sample.
 169. A method ofanalyzing a protein sample, the method comprising (a) treating a proteinsample to produce protein fragments, wherein the protein sample has aknown amount of protein, wherein the protein sample comprises a targetprotein, wherein the protein fragments comprise a target proteinfragment derived from the target protein, (b) mixing the protein samplewith a predetermined amount of a reporter signal calibrator, wherein thetarget protein fragment can be altered, wherein the reporter signalcalibrator can be altered, wherein the altered form of the reportersignal calibrator can be distinguished from the altered form of thetarget protein fragment, (b) altering the target protein fragment andreporter signal calibrator, (c) detecting the altered forms of thetarget protein fragment and reporter signal calibrator.
 170. The methodof claim 169 further comprising determining the ratio of the amount ofthe target protein fragment and the amount of the reporter signalcalibrator detected, and comparing the determined ratio with thepredicted ratio of the amount of the target protein fragment and theamount of the reporter signal calibrator, wherein the predicted ratio isbased on the predicted amount of target protein fragment in the proteinsample and the predetermined amount of reporter signal calibrator,wherein the predicted amount of target protein fragment is the amount oftarget protein fragment the protein sample would have if the knownamount of protein in the protein sample consisted of the target protein,wherein the difference between the determined ratio and the predictedratio is a measure of the purity of the protein sample for the targetprotein, wherein the closer the determined ratio is to the predictedratio, the purer the protein sample.
 171. A set of reporter signalcalibrators, wherein each reporter signal calibrator shares a commonproperty with a target protein fragment in a set of target proteinfragments, wherein the common property allows the target proteinfragments and reporter signal calibrators having the common property tobe distinguished or separated from molecules lacking the commonproperty, wherein the target protein fragment and reporter signalcalibrator that share a common property correspond to each other,wherein the target protein fragments can be altered, wherein the alteredforms of the target protein fragments can be distinguished from theother altered forms of the target protein fragments, wherein thereporter signal calibrators can be altered, wherein the altered form ofeach reporter signal calibrator can be distinguished from the alteredform of the target protein fragment with which the reporter signalcalibrator shares a common property.
 172. The set of claim 171 whereinthe set includes a predetermined amount of each reporter signalcalibrator.
 173. The set of claim 172 wherein the amount of at least twoof the reporter signal calibrators is different.
 174. The set of claim172 wherein the relative amount each reporter signal calibrator is basedon the relative amount of each corresponding target protein fragmentexpected to be in the protein sample.
 175. The set of claim 172 whereinthe amount of each of the reporter signal calibrators is the same. 176.A kit for producing a protein signature, the kit comprising (a) a set ofreporter signal calibrators, wherein each reporter signal calibratorshares a common property with a target protein fragment in a set oftarget protein fragments, wherein the common property allows the targetprotein fragments and reporter signal calibrators having the commonproperty to be distinguished or separated from molecules lacking thecommon property, wherein the target protein fragment and reporter signalcalibrator that share a common property correspond to each other,wherein the target protein fragments can be altered, wherein the alteredforms of the target protein fragments can be distinguished from theother altered forms of the target protein fragments, wherein thereporter signal calibrators can be altered, wherein the altered form ofeach reporter signal calibrator can be distinguished from the alteredform of the target protein fragment with which the reporter signalcalibrator shares a common property, (b) one or more reagents fortreating a protein sample to produce protein fragments.
 177. A mixturecomprising a set of reporter signal calibrators and a set of targetprotein fragments, wherein each reporter signal calibrator shares acommon property with a target protein fragment in the set of targetprotein fragments, wherein the common property allows the target proteinfragments and reporter signal calibrators having the common property tobe distinguished or separated from molecules lacking the commonproperty, wherein the target protein fragment and reporter signalcalibrator that share a common property correspond to each other,wherein the target protein fragments can be altered, wherein the alteredforms of the target protein fragments can be distinguished from theother altered forms of the target protein fragments, wherein thereporter signal calibrators can be altered, wherein the altered form ofeach reporter signal calibrator can be distinguished from the alteredform of the target protein fragment with which the reporter signalcalibrator shares a common property.
 178. A set of target proteinfragments, wherein each target protein fragment shares a common propertywith a reporter signal calibrator in a set of reporter signalcalibrators, wherein the common property allows the target proteinfragments and reporter signal calibrators having the common property tobe distinguished or separated from molecules lacking the commonproperty, wherein the target protein fragment and reporter signalcalibrator that share a common property correspond to each other,wherein the target protein fragments can be altered, wherein the alteredforms of the target protein fragments can be distinguished from theother altered forms of the target protein fragments, wherein thereporter signal calibrators can be altered, wherein the altered form ofeach reporter signal calibrator can be distinguished from the alteredform of the target protein fragment with which the reporter signalcalibrator shares a common property.
 179. A method of producing aprotein signature, the method comprising (a) treating a protein sampleto produce protein fragments, wherein the protein fragments comprise aset of target protein fragments, wherein each of the target proteinfragments can be altered, wherein the altered forms of each targetprotein fragment can be distinguished from every other altered form oftarget protein fragment, (b) mixing the target protein fragments with aset of reporter signal calibrators, wherein each target protein fragmentshares a common property with at least one of the reporter signalcalibrators, wherein the common property allows each of the targetprotein fragments and reporter signal calibrators having the commonproperty to be distinguished or separated from molecules lacking thecommon property, wherein the target protein fragment and reporter signalcalibrator that share a common property correspond to each other,wherein each of the reporter signal calibrators can be altered, whereinthe altered form of each reporter signal calibrator can be distinguishedfrom the altered form of the target protein fragment with which thereporter signal calibrator shares a common property, (c) separating thetarget protein fragments and reporter signal calibrators from othermolecules based on the common properties of the target protein fragmentsand reporter signal calibrators, (d) altering the target proteinfragments and reporter signal calibrators, (e) detecting the alteredforms of the target protein fragments and reporter signal calibrators,wherein the presence, absence, amount, or presence and amount of thealtered forms of the target protein fragments indicates the presence,absence, amount, or presence and amount in the protein sample of thetarget protein fragments from which the altered forms of the targetprotein fragments are derived, wherein the presence, absence, amount, orpresence and amount of the target protein fragments in the proteinsample constitutes a protein signature of the protein sample.
 180. Amethod of producing a protein signature, the method comprising detectingaltered forms of target protein fragments and reporter signalcalibrators, wherein the altered forms of each target protein fragmentcan be distinguished from every other altered form of target proteinfragment, wherein each target protein fragment shares a common propertywith at least one of the reporter signal calibrators, wherein the commonproperty allows each of the target protein fragments and reporter signalcalibrators having the common property to be distinguished or separatedfrom molecules lacking the common property, wherein the target proteinfragment and reporter signal calibrator that share a common propertycorrespond to each other, wherein the altered form of each reportersignal calibrator can be distinguished from the altered form of thetarget protein fragment with which the reporter signal calibrator sharesa common property, wherein the presence, absence, amount, or presenceand amount of the altered forms of the target protein fragmentsindicates the presence, absence, amount, or presence and amount in aprotein sample of the target protein fragments from which the alteredforms of the target protein fragments are derived, wherein the presence,absence, amount, or presence and amount of the target protein fragmentsin the protein sample constitutes a protein signature of the proteinsample.
 181. A method of producing a protein signature, the methodcomprising (a) treating a protein sample to produce protein fragments,wherein the protein fragments comprise a set of target proteinfragments, wherein each of the target protein fragments can be altered,wherein the altered forms of each target protein fragment can bedistinguished from every other altered form of target protein fragment,(b) separating the target protein fragments from other protein fragmentsin the protein sample, (c) altering the target protein fragments, (d)detecting the altered forms of the target protein fragments, wherein thepresence, absence, amount, or presence and amount of the altered formsof the target protein fragments indicates the presence, absence, amount,or presence and amount in the protein sample of the target proteinfragments from which the altered forms of the target protein fragmentsare derived, wherein the presence, absence, amount, or presence andamount of the target protein fragments in the protein sample constitutesa protein signature of the protein sample.
 182. The method of claim 181further comprising, prior to or simultaneous with step (b), mixing thetarget protein fragments with a set of reporter signal calibrators,wherein each target protein fragment shares a common property with atleast one of the reporter signal calibrators, wherein the commonproperty allows each of the target protein fragments and reporter signalcalibrators having the common property to be distinguished or separatedfrom molecules lacking the common property, wherein each of the reportersignal calibrators can be altered, wherein the altered form of eachreporter signal calibrator can be distinguished from the altered form ofthe target protein fragment with which the reporter signal calibratorshares a common property.
 183. A set of reporter signal calibrators,wherein each reporter signal calibrator shares a common property with atarget protein fragment in a set of target protein fragments, whereinthe common property allows each of the target protein fragments andreporter signal calibrators having the common property to bedistinguished or separated from molecules lacking the common property,wherein the target protein fragment and reporter signal calibrator thatshare a common property correspond to each other, wherein each of thetarget protein fragments can be altered, wherein the altered forms ofeach target protein fragment can be distinguished from every otheraltered form of target protein fragment, wherein each of the reportersignal calibrators can be altered, wherein the altered form of eachreporter signal calibrator can be distinguished from the altered form ofthe target protein fragment with which the reporter signal calibratorshares a common property.
 184. A kit for producing a protein signature,the kit comprising (a) a set of reporter signal calibrators, whereineach reporter signal calibrator shares a common property with a targetprotein fragment in a set of target protein fragments, wherein thecommon property allows each of the target protein fragments and reportersignal calibrators having the common property to be distinguished orseparated from molecules lacking the common property, wherein the targetprotein fragment and reporter signal calibrator that share a commonproperty correspond to each other, wherein each of the target proteinfragments can be altered, wherein the altered forms of each targetprotein fragment can be distinguished from every other altered form oftarget protein fragment, wherein each of the reporter signal calibratorscan be altered, wherein the altered form of each reporter signalcalibrator can be distinguished from the altered form of the targetprotein fragment with which the reporter signal calibrator shares acommon property, (b) one or more reagents for treating a protein sampleto produce protein fragments.
 185. A mixture comprising a set ofreporter signal calibrators and a set of target protein fragments,wherein each reporter signal calibrator shares a common property with atarget protein fragment in the set of target protein fragments, whereinthe common property allows each of the target protein fragments andreporter signal calibrators having the common property to bedistinguished or separated from molecules lacking the common property,wherein the target protein fragment and reporter signal calibrator thatshare a common property correspond to each other, wherein each of thetarget protein fragments can be altered, wherein the altered forms ofeach target protein fragment can be distinguished from every otheraltered form of target protein fragment, wherein each of the reportersignal calibrators can be altered, wherein the altered form of eachreporter signal calibrator can be distinguished from the altered form ofthe target protein fragment with which the reporter signal calibratorshares a common property.
 186. A set of target protein fragments,wherein each target protein fragment shares a common property with areporter signal calibrator in a set of reporter signal calibrators,wherein the common property allows each of the target protein fragmentsand reporter signal calibrators having the common property to bedistinguished or separated from molecules lacking the common property,wherein the target protein fragment and reporter signal calibrator thatshare a common property correspond to each other, wherein each of thetarget protein fragments can be altered, wherein the altered forms ofeach target protein fragment can be distinguished from every otheraltered form of target protein fragment, wherein each of the reportersignal calibrators can be altered, wherein the altered form of eachreporter signal calibrator can be distinguished from the altered form ofthe target protein fragment with which the reporter signal calibratorshares a common property.
 187. A set of nucleic acid molecules whereineach nucleic acid molecule comprises a nucleotide segment encoding anamino acid segment comprising a reporter signal peptide and a protein orpeptide of interest, wherein the reporter signal peptides have a commonproperty, wherein the common property allows the reporter signalpeptides to be distinguished or separated from molecules lacking thecommon property, wherein the reporter signal peptides can be altered,wherein the altered form of each reporter signal peptide can bedistinguished from the altered forms of the other reporter signalpeptides.
 188. The set of claim 187 wherein each nucleic acid moleculefurther comprises expression sequences, wherein the expression sequencesare operably linked to the nucleotide segment such that the amino acidsegment can be expressed.
 189. The set of claim 188 wherein theexpression sequences comprise translation expression sequences.
 190. Theset of claim 189 wherein the expression sequences further comprisetranscription expression sequences.
 191. The set of claim 188 whereinthe amino acid segment can be expressed in vitro.
 192. The set of claim188 wherein the amino acid segment can be expressed in vivo.
 193. Theset of claim 192 wherein the amino acid segment can be expressed in cellculture.
 194. The set of claim 188 wherein the expression sequences ofeach nucleic acid molecule are different.
 195. The set of claim 194wherein the different expression sequences are differently regulated.196. The set of claim 194 wherein the expression sequences are similarlyregulated.
 197. The set of claim 196 wherein a plurality of theexpression sequences are expression sequences of, or derived from, genesexpressed as part of the same expression cascade.
 198. The set of claim188 wherein the expression sequences of each nucleic acid molecule arethe same.
 199. The set of claim 198 wherein the expression sequences aresimilarly regulated.
 200. The set of claim 188 wherein the expressionsequences of at least two nucleic acid molecules are different.
 201. Theset of claim 188 wherein the expression sequences of at least twonucleic acid molecules are the same.
 202. The set of claim 187 whereineach nucleic acid molecule further comprises replication sequences,wherein the replication sequences allow replication of the nucleic acidmolecules.
 203. The set of claim 202 wherein the nucleic acid moleculescan be replicated in vitro.
 204. The set of claim 202 wherein thenucleic acid molecules can be replicated in vivo.
 205. The set of claim204 wherein the nucleic acid molecules can be replicated in cellculture.
 206. The set of claim 187 wherein each nucleic acid moleculefurther comprises integration sequences, wherein the integrationsequences allow integration of the nucleic acid molecules into othernucleic acids.
 207. The set of claim 206 wherein the nucleic acidmolecules can be integrated into a chromosome.
 208. The set of claim 207wherein the nucleic acid molecules can be integrated into a chromosomeat a predetermined location.
 209. The set of claim 187 wherein thenucleic acids molecules are produced by replicating nucleic acids in oneor more nucleic acid samples.
 210. The set of claim 209 wherein thenucleic acids are replicated using pairs of primers, wherein each of thefirst primers in the primer pairs used to produce the nucleic acidmolecules comprises a nucleotide sequence encoding the reporter signalpeptide.
 211. The set of claim 210 wherein each first primer furthercomprises expression sequences.
 212. The set of claim 211 wherein thenucleotide sequence of each first primer also encodes an epitope tag.213. The set of claim 187 wherein each amino acid segment furthercomprises an epitope tag.
 214. The set of claim 213 wherein the epitopetag of each amino acid segment is different.
 215. The set of claim 213wherein the epitope tag of each amino acid segment is the same.
 216. Theset of claim 213 wherein the epitope tag of at least two amino acidsegments are different.
 217. The set of claim 213 wherein the epitopetag of at least two amino acid segments are the same.
 218. The set ofclaim 187 wherein the reporter signal peptide of each amino acid segmentis different.
 219. The set of claim 187 wherein the reporter signalpeptide of each amino acid segment is the same.
 220. The set of claim187 wherein the reporter signal peptide of at least two amino acidsegments are different.
 221. The set of claim 187 wherein the reportersignal peptide of at least two amino acid segments are the same. 222.The set of claim 187 wherein the nucleic acid molecules are in cells.223. The set of claim 222 wherein each nucleic acid molecule is in adifferent cell.
 224. The set of claim 222 wherein each nucleic acidmolecule is in the same cell.
 225. The set of claim 224 wherein eachnucleic acid molecule further comprises expression sequences, whereinthe expression sequences are operably linked to the nucleotide segmentsuch that the amino acid segment can be expressed.
 226. The set of claim225 wherein the expression sequences of each nucleic acid molecule aredifferent.
 227. The set of claim 226 wherein the expression sequencesare similarly regulated.
 228. The set of claim 227 wherein a pluralityof the expression sequences are expression sequences of, or derivedfrom, genes expressed as part of the same expression cascade.
 229. Theset of claim 222 wherein the nucleic acid molecules are integrated intoa chromosome of the cell.
 230. The set of claim 229 wherein the nucleicacid molecules are integrated into the chromosome at a predeterminedlocation.
 231. The set of claim 229 wherein the chromosome is anartificial chromosome.
 232. The set of claim 222 wherein the nucleicacid molecules are, or are integrated into, a plasmid.
 233. The set ofclaim 222 wherein the cells are in cell lines.
 234. The set of claim 233wherein each nucleic acid molecule is in a different cell line.
 235. Theset of claim 233 wherein each nucleic acid molecule is in the same cellline.
 236. The set of claim 187 wherein the nucleic acid molecules arein organisms.
 237. The set of claim 236 wherein each nucleic acidmolecule is in a different organism.
 238. The set of claim 236 whereineach nucleic acid molecule is in the same organism.
 239. The set ofclaim 238 wherein each nucleic acid molecule further comprisesexpression sequences, wherein the expression sequences are operablylinked to the nucleotide segment such that the amino acid segment can beexpressed.
 240. The set of claim 239 wherein the expression sequences ofeach nucleic acid molecule are different.
 241. The set of claim 240wherein the expression sequences are similarly regulated.
 242. The setof claim 241 wherein a plurality of the expression sequences areexpression sequences of, or derived from, genes expressed as part of thesame expression cascade.
 243. The set of claim 236 wherein the nucleicacid molecules are integrated into a chromosome of the organism. 244.The set of claim 243 wherein the nucleic acid molecules are integratedinto the chromosome at a predetermined location.
 245. The set of claim243 wherein the chromosome is an artificial chromosome.
 246. The set ofclaim 236 wherein the nucleic acid molecules are, or are integratedinto, a plasmid.
 247. The set of claim 236 wherein each nucleic acidmolecule is in a different organism.
 248. The set of claim 236 whereineach nucleic acid molecule is in the same organism.
 249. The set ofclaim 187 wherein the nucleic acid molecules are in cells of anorganism.
 250. The set of claim 249 wherein the nucleic acid moleculesare in substantially all of the cells of the organism.
 251. The set ofclaim 249 wherein the nucleic acid molecules are in some of the cells ofthe organism.
 252. The set of claim 249 wherein the amino acid segmentsare expressed in substantially all of the cells of the organism. 253.The set of claim 249 wherein the amino acid segments are expressed insome of the cells of the organism.
 254. The set of claim 187 wherein theprotein or peptide of interest of each amino acid segment is different.255. The set of claim 187 wherein the protein or peptide of interest ofeach amino acid segment is the same.
 256. The set of claim 187 whereinthe protein or peptide of interest of at least two amino acid segmentsare different.
 257. The set of claim 187 wherein the protein or peptideof interest of at least two amino acid segments are the same.
 258. Theset of claim 254 wherein the proteins or peptides of interest arerelated.
 259. The set of claim 258 wherein the proteins or peptides ofinterest are proteins produced in the same cascade.
 260. The set ofclaim 258 wherein the proteins or peptides of interest are proteinsexpressed under the same conditions.
 261. The set of claim 258 whereinthe proteins or peptides of interest are proteins associated with thesame disease.
 262. The set of claim 258 wherein the proteins or peptidesof interest are proteins associated with the same cell type.
 263. Theset of claim 258 wherein the proteins or peptides of interest areproteins associated with the same tissue type.
 264. The set of claim 258wherein the proteins or peptides of interest are proteins in the sameenzymatic pathway.
 265. The set of claim 187 wherein the nucleotidesegment encodes a plurality of amino acid segments each comprising areporter signal peptide and a protein or peptide of interest.
 266. Theset of claim 265 wherein the protein or peptide of interest of at leasttwo of the amino acid segments in one of the nucleotide segments aredifferent.
 267. The set of claim 265 wherein the protein or peptide ofinterest of the amino acid segments in one of the nucleotide segmentsare different.
 268. The set of claim 265 wherein the protein or peptideof interest of at least two of the amino acid segments in each of thenucleotide segments are different.
 269. The set of claim 265 wherein theprotein or peptide of interest of the amino acid segments in each of thenucleotide segments are different.
 270. The set of claim 265 wherein theset consists of a single nucleic acid molecule.
 271. The set of claim187 wherein the set consists of a single nucleic acid molecule, whereinthe nucleic acid molecule comprises a plurality of nucleotide segmentseach encoding an amino acid segment.
 272. The set of claim 187 whereinthe amino acid segment comprises a cleavage site near the junctionbetween the reporter signal peptide and the protein or peptide ofinterest.
 273. The set of claim 272 wherein the cleavage site is atrypsin cleavage site.
 274. The set of claim 272 wherein the cleavagesite is at the junction between the reporter signal peptide and theprotein or peptide of interest.
 275. The set of claim 187 wherein eachamino acid segment further comprises a self-cleaving segment.
 276. Theset of claim 275 wherein the self-cleaving segment is between thereporter signal peptide and the protein or peptide of interest.
 277. Theset of claim 275 wherein the self-cleaving segment is an intein segment.278. A set of nucleic acid molecules wherein each nucleic acid moleculecomprises a nucleotide segment encoding an amino acid segment comprisinga reporter signal peptide and a protein or peptide of interest, whereinthe reporter signal peptides have a common property, wherein the commonproperty allows the reporter signal peptides to be distinguished orseparated from molecules lacking the common property, wherein thereporter signal peptides can be altered, wherein alteration of thereporter signal peptides alters the amino acid segments, wherein thealtered form of each amino acid segment can be distinguished from thealtered forms of the other amino acid segments.
 279. A set of nucleicacid molecules wherein each nucleic acid molecule comprises a nucleotidesegment encoding an amino acid segment comprising a reporter signalpeptide and a protein or peptide of interest, wherein the amino acidsegments have a common property, wherein the common property allows theamino acid segments to be distinguished or separated from moleculeslacking the common property, wherein the reporter signal peptides can bealtered, wherein the altered form of each reporter signal peptide can bedistinguished from the altered forms of the other reporter signalpeptides.
 280. A set of nucleic acid molecules wherein each nucleic acidmolecule comprises a nucleotide segment encoding an amino acid segmentcomprising a reporter signal peptide and a protein or peptide ofinterest, wherein the amino acid segments have a common property,wherein the common property allows the amino acid segments to bedistinguished or separated from molecules lacking the common property,wherein the reporter signal peptides can be altered, wherein alterationof the reporter signal peptides alters the amino acid segments, whereinthe altered form of each amino acid segment can be distinguished fromthe altered forms of the other amino acid segments.
 281. A set ofnucleic acid molecules wherein each nucleic acid molecule comprises anucleotide segment encoding an amino acid segment comprising a reportersignal peptide and a protein or peptide of interest, wherein the aminoacid segments each comprise an amino acid subsegment, wherein each aminoacid subsegment comprises a portion of the protein or peptide ofinterest and all or a portion of the reporter signal peptide, whereinthe amino acid subsegments have a common property, wherein the commonproperty allows the amino acid subsegments to be distinguished orseparated from molecules lacking the common property, wherein thereporter signal peptides can be altered, wherein the altered form ofeach reporter signal peptide can be distinguished from the altered formsof the other reporter signal peptides.
 282. A set of nucleic acidmolecules wherein each nucleic acid molecule comprises a nucleotidesegment encoding an amino acid segment comprising a reporter signalpeptide and a protein or peptide of interest, wherein the amino acidsegments each comprise an amino acid subsegment, wherein each amino acidsubsegment comprises a portion of the protein or peptide of interest andall or a portion of the reporter signal peptide, wherein the amino acidsubsegments have a common property, wherein the common property allowsthe amino acid subsegments to be distinguished or separated frommolecules lacking the common property, wherein the reporter signalpeptides can be altered, wherein alteration of the reporter signalpeptides alters the amino acid subsegments, wherein the altered form ofeach amino acid subsegment can be distinguished from the altered formsof the other amino acid subsegments.
 283. A set of amino acid segmentswherein each amino acid segment comprises a reporter signal peptide anda protein or peptide of interest, wherein the reporter signal peptideshave a common property, wherein the common property allows the reportersignal peptides to be distinguished or separated from molecules lackingthe common property, wherein the reporter signal peptides can bealtered, wherein the altered form of each reporter signal peptide can bedistinguished from the altered forms of the other reporter signalpeptides.
 284. The set of claim 283 wherein the amino acid segment is aprotein or peptide.
 285. The set of claim 283 wherein the set consistsof a single amino acid segment, wherein the amino acid segment comprisesa plurality of reporter signal peptides.
 286. A cell comprising a set ofnucleic acid molecules wherein each nucleic acid molecule comprises anucleotide segment encoding an amino acid segment comprising a reportersignal peptide and a protein or peptide of interest, wherein thereporter signal peptides have a common property, wherein the commonproperty allows the reporter signal peptides to be distinguished orseparated from molecules lacking the common property, wherein thereporter signal peptides can be altered, wherein the altered form ofeach reporter signal peptide can be distinguished from the altered formsof the other reporter signal peptides.
 287. A set of cells wherein eachcell comprises a nucleic acid molecule wherein each nucleic acidmolecule comprises a nucleotide segment encoding an amino acid segmentcomprising a reporter signal peptide and a protein or peptide ofinterest, wherein the reporter signal peptides have a common property,wherein the common property allows the reporter signal peptides to bedistinguished or separated from molecules lacking the common property,wherein the reporter signal peptides can be altered, wherein the alteredform of each reporter signal peptide can be distinguished from thealtered forms of the other reporter signal peptides.
 288. The set ofclaim 287 wherein each cell further comprises additional nucleic acidmolecules.
 289. The set of claim 287 wherein the set consists of asingle cell, wherein the cell comprises a plurality of nucleic acidmolecules.
 290. The set of claim 287 wherein the set consists of asingle cell, wherein the cell comprises a set of nucleic acid molecules,wherein the set of nucleic acid molecules consists of a single nucleicacid molecule, wherein the nucleic acid molecule encodes a plurality ofnucleic acid segments.
 291. An organism comprising a set of nucleic acidmolecules wherein each nucleic acid molecule comprises a nucleotidesegment encoding an amino acid segment comprising a reporter signalpeptide and a protein or peptide of interest, wherein the reportersignal peptides have a common property, wherein the common propertyallows the reporter signal peptides to be distinguished or separatedfrom molecules lacking the common property, wherein the reporter signalpeptides can be altered, wherein the altered form of each reportersignal peptide can be distinguished from the altered forms of the otherreporter signal peptides.
 292. A set of organisms each organismcomprises a nucleic acid molecule wherein each nucleic acid moleculecomprises a nucleotide segment encoding an amino acid segment comprisinga reporter signal peptide and a protein or peptide of interest, whereinthe reporter signal peptides have a common property, wherein the commonproperty allows the reporter signal peptides to be distinguished orseparated from molecules lacking the common property, wherein thereporter signal peptides can be altered, wherein the altered form ofeach reporter signal peptide can be distinguished from the altered formsof the other reporter signal peptides.
 293. The set of claim 292 whereineach organism further comprises additional nucleic acid molecules. 294.The set of claim 292 wherein the set consists of a single organism,wherein the organism comprises a plurality of nucleic acid molecules.295. The set of claim 292 wherein the set consists of a single organism,wherein the organism comprises a set of nucleic acid molecules, whereinthe set of nucleic acid molecules consists of a single nucleic acidmolecule, wherein the nucleic acid molecule encodes a plurality ofnucleic acid segments.
 296. A method of detecting expression, the methodcomprising detecting a target altered reporter signal peptide derivedfrom one or more expression samples, wherein the one or more expressionsamples collectively comprise a set of nucleic acid molecules, whereineach nucleic acid molecule comprises a nucleotide segment encoding anamino acid segment comprising a reporter signal peptide and a protein orpeptide of interest, wherein the reporter signal peptides have a commonproperty, wherein the common property allows the reporter signalpeptides to be distinguished or separated from molecules lacking thecommon property, wherein the reporter signal peptides can be altered,wherein the altered form of each reporter signal peptide can bedistinguished from the altered forms of the other reporter signalpeptides, wherein the target altered reporter signal peptide is one ofthe altered reporter signal peptides, wherein detection of the targetaltered reporter signal peptide indicates expression of the amino acidsegment that comprises the reporter signal peptide from which the targetaltered reporter signal peptide is derived.
 297. The method of claim 296further comprising determining the amount of the target altered reportersignal peptide detected, wherein the amount of the target alteredreporter signal peptide indicates the amount present in the one or moreexpression samples of the amino acid segment that comprises the reportersignal peptide from which the target altered reporter signal peptide isderived.
 298. The method of claim 297 wherein the amount of the aminoacid segment present is proportional to the amount of the target alteredreporter signal peptide detected.
 299. The method of claim 296 furthercomprising detecting a plurality of the altered reporter signalpeptides, wherein detection of each altered reporter signal peptideindicates expression of the amino acid segment that comprises thereporter signal peptide from which that altered reporter signal peptideis derived.
 300. The method of claim 299 further comprising determiningthe amount of the altered reporter signal peptides detected, wherein theamount of each altered reporter signal peptide indicates the amountpresent in the one or more expression samples of the amino acid segmentthat comprises the reporter signal peptide from which that alteredreporter signal peptide is derived.
 301. The method of claim 300 whereinthe amount of the amino acid segment present is proportional to theamount of the altered reporter signal peptide detected.
 302. The methodof claim 299 wherein the presence, absence, amount, or presence andamount of the altered forms of the reporter signal peptides indicatesthe presence, absence, amount, or presence and amount in the expressionsample of the reporter signal peptides from which the altered forms ofthe reporter signal peptides are derived, wherein the presence, absence,amount, or presence and amount of the reporter signal peptides in theexpression sample constitutes a protein signature of the expressionsample.
 303. The method of claim 302 wherein the altered forms of thereporter signal peptides are detecting using mass spectrometry.
 304. Themethod of claim 303 wherein the altered forms of the reporter signalpeptides are detected with a tandem mass spectrometer.
 305. The methodof claim 304 wherein the mass spectrometer includes a quadrupole set forsingle-ion filtering, a collision cell, and a time-of-flightspectrometer.
 306. The method of claim 302 wherein the reporter signalpeptides are altered by fragmentation.
 307. The method of claim 306wherein the reporter signal peptides are altered by cleavage at aphotocleavable amino acid.
 308. The method of claim 306 wherein thereporter signal peptides are fragmented in a collision cell.
 309. Themethod of claim 306 wherein the reporter signal peptides are fragmentedat an asparagine-proline bond, a methionine, or a phosphorylated aminoacid.
 310. The method of claim 302 wherein the common property ismass-to-charge ratio, wherein the reporter signal peptides are alteredby altering their mass, their charge, or their mass and charge, whereinthe altered forms of the reporter signal peptides can be distinguishedvia differences in the mass-to-charge ratio of the altered forms of thereporter signal peptides.
 311. The method of claim 302 wherein there aretwo or more, three or more, four or more, five or more, six or more,seven or more, eight or more, nine or more, ten or more, twenty or more,thirty or more, forty or more, fifty or more, sixty or more, seventy ormore, eighty or more, ninety or more, or one hundred or more differentreporter signal peptides.
 312. The method of claim 311 wherein there areten or more different reporter signal peptides.
 313. The method of claim312 wherein each reporter signal peptide has a labile or scissile bondin a different location.
 314. The method of claim 302 further comprisingcomparing the protein signature to one or more other protein signatures.315. The method of claim 302 wherein the detected altered reportersignal peptides are derived from a plurality of expression samples. 316.The method of claim 315 wherein some of the detected altered reportersignal peptides derived from a control expression sample, identifyingdifferences between the protein signatures produced from the expressionsamples and the control expression sample.
 317. The method of claim 316wherein the differences are differences in the presence, amount,presence and amount, or absence of reporter signal peptides in theexpression samples and the control expression sample.
 318. The method ofclaim 315 wherein the plurality of expression samples comprises acontrol expression sample and a tester expression sample, wherein thetester expression sample, or the source of the tester expression sample,is treated so as to destroy, disrupt or eliminate one or more of theamino acid segments in the tester expression sample, wherein thereporter signal peptides corresponding to the destroyed, disrupted, oreliminated amino acid segments will be produced from the controlexpression sample but not the tester expression sample.
 319. The methodof claim 318 wherein the tester expression sample is treated so as todestroy, disrupt or eliminate one or more of the amino acid segments inthe tester expression sample.
 320. The method of claim 319 wherein oneor more of the amino acid segments in the tester sample are eliminatedby separating the one or more of the amino acid segments from the testerexpression sample.
 321. The method of claim 320 wherein the one or moreof the amino acid segments are separated by affinity separation. 322.The method of claim 318 wherein the source of the tester expressionsample is treated so as to destroy, disrupt or eliminate one or more ofthe amino acid segments in the tester expression sample.
 323. The methodof claim 322 wherein the treatment of the source is accomplished byexposing cells from which the tester sample will be derived with acompound, composition, or condition that will reduce or eliminateexpression of one or more of the nucleotide segments.
 324. The method ofclaim 318 further comprising identifying differences in the reportersignal peptides in the control expression sample and tester expressionsample.
 325. The method of claim 315 further comprising identifyingdifferences between the reporter signal peptides in the expressionsamples.
 326. The method of claim 315 wherein at least two of theexpression samples, or the sources of the at least two expressionsamples, are subjected to different conditions.
 327. The method of claim326 wherein the sources of the expression samples are cells.
 328. Themethod of claim 326 wherein differences in the protein signatures of theat least two expression samples indicate the effect of the differentconditions.
 329. The method of claim 326 wherein the differentconditions are exposure to different compounds.
 330. The method of claim326 wherein the different conditions are exposure to a compound and noexposure to the compound.
 331. The method of claim 302 furthercomprising producing a second protein signature from a second expressionsample and comparing the first protein signature and second proteinsignature, wherein differences in the first and second proteinsignatures indicate differences in source or condition of the source ofthe first and second expression samples.
 332. The method of claim 302further comprising producing a second protein signature from a secondexpression sample and comparing the first protein signature and secondprotein signature, wherein differences in the first and second proteinsignatures indicate differences in protein modification of the first andsecond expression samples.
 333. The method of claim 332 wherein thesecond expression sample is a sample from the same type of cells as thefirst expression sample except that the cells from which the firstexpression sample is derived are modification-deficient relative to thecells from which the second expression sample is derived.
 334. Themethod of claim 332 wherein the second expression sample is a samplefrom a different type of cells than the first expression sample, andwherein the cells from which the first expression sample is derived aremodification-deficient relative to the cells from which the secondexpression sample is derived.
 335. The method of claim 302 wherein theexpression sample is derived from one or more cells.
 336. The method ofclaim 335 wherein the protein signature indicates the physiologicalstate of the cells.
 337. The method of claim 335 wherein the proteinsignature indicates the effect of a treatment of the cells.
 338. Themethod of claim 337 wherein the cells are derived from an organism,wherein the cells are treated by treating the organism.
 339. The methodof claim 338 wherein the organism is treated by administering a compoundto the organism.
 340. The method of claim 338 wherein the organism ishuman.
 341. The method of claim 299 wherein altered reporter signalpeptides are detected in a first and a second expression sample. 342.The method of claim 341 wherein the second expression sample is a samplefrom the same type of organism as the first expression sample.
 343. Themethod of claim 341 wherein the second expression sample is a samplefrom the same type of tissue as the first expression sample.
 344. Themethod of claim 341 wherein the second expression sample is a samplefrom the same organism as the first expression sample.
 345. The methodof claim 344 wherein the second expression sample is obtained at adifferent time than the first expression sample.
 346. The method ofclaim 341 wherein the second expression sample is a sample from adifferent organism than the first expression sample.
 347. The method ofclaim 341 wherein the second expression sample is a sample from adifferent type of tissue than the first expression sample.
 348. Themethod of claim 341 wherein the second expression sample is a samplefrom a different species of organism than the first expression sample.349. The method of claim 341 wherein the second expression sample is asample from a different strain of organism than the first expressionsample.
 350. The method of claim 341 wherein the second expressionsample is a sample from a different cellular compartment than the firstexpression sample.
 351. The method of claim 296 further comprisingaltering the reporter signal peptides.
 352. The method of claim 351wherein the reporter signal peptides are altered by fragmentation. 353.The method of claim 352 wherein the reporter signal peptides are alteredby cleavage at a photocleavable amino acid.
 354. The method of claim 352wherein the reporter signal peptides are fragmented in a collision cell.355. The method of claim 352 wherein the reporter signal peptides arefragmented at an asparagine-proline bond, a methionine, or aphosphorylated amino acid.
 356. The method of claim 296 furthercomprising separating the reporter signal peptides from the expressionsamples.
 357. The method of claim 356 wherein the reporter signalpeptides are distinguished or separated from the expression samplesbased on the common property.
 358. The method of claim 296 furthercomprising cleaving the reporter signal peptides from the proteins orpeptides of interest.
 359. The method of claim 358 wherein the reportersignal peptides are distinguished or separated from the proteins orpeptides of interest based on the common property.
 360. The method ofclaim 296 further comprising cleaving the amino acid segments into areporter signal peptide portion and a protein portion.
 361. The methodof claim 296 further comprising mixing two or more of the expressionsamples together.
 362. The method of claim 296 further comprising mixingtwo or more amino acid segments together, wherein the mixed amino acidsegments were derived from two or more different expression samples.363. The method of claim 296 wherein expression of the amino acidsegment that comprises the reporter signal peptide from which the targetaltered reporter signal peptide is derived identifies the expressionsample from which the target altered reporter signal peptide is derived.364. The method of claim 363 wherein the expression samples are derivedfrom one or more cells, wherein expression of the amino acid segmentthat comprises the reporter signal peptide from which the target alteredreporter signal peptide is derived identifies the cell from which theidentified expression sample is derived.
 365. The method of claim 363wherein the expression samples are derived from one or more organisms,wherein expression of the amino acid segment that comprises the reportersignal peptide from which the target altered reporter signal peptide isderived identifies the organism from which the identified expressionsample is derived.
 366. The method of claim 363 wherein the expressionsamples are derived from one or more tissues, wherein expression of theamino acid segment that comprises the reporter signal peptide from whichthe target altered reporter signal peptide is derived identifies thetissue from which the identified expression sample is derived.
 367. Themethod of claim 363 wherein the expression samples are derived from oneor more cell lines, wherein expression of the amino acid segment thatcomprises the reporter signal peptide from which the target alteredreporter signal peptide is derived identifies the cell line from whichthe identified expression sample is derived.
 368. The method of claim296 wherein each nucleic acid molecule further comprises expressionsequences, wherein the expression sequences are operably linked to thenucleotide segment such that the amino acid segment is expressed. 369.The method of claim 368 wherein the expression sequences comprisetranslation expression sequences.
 370. The method of claim 369 whereinthe expression sequences further comprise transcription expressionsequences.
 371. The method of claim 368 wherein the amino acid segmentis expressed in vitro.
 372. The method of claim 368 wherein the aminoacid segment is expressed in vivo.
 373. The method of claim 372 whereinthe amino acid segment is expressed in cell culture.
 374. The method ofclaim 368 wherein the expression sequences of each nucleic acid moleculeare different.
 375. The method of claim 374 wherein the differentexpression sequences are differently regulated.
 376. The method of claim374 wherein the expression sequences are similarly regulated.
 377. Themethod of claim 376 wherein a plurality of the expression sequences areexpression sequences of, or derived from, genes expressed as part of thesame expression cascade.
 378. The method of claim 368 wherein theexpression sequences of each nucleic acid molecule are the same. 379.The method of claim 378 wherein the expression sequences are similarlyregulated.
 380. The method of claim 368 wherein the expression sequencesof at least two nucleic acid molecules are different.
 381. The method ofclaim 368 wherein the expression sequences of at least two nucleic acidmolecules are the same.
 382. The method of claim 368 wherein expressionof the amino acid segment is induced.
 383. The method of claim 296wherein each nucleic acid molecule further comprises replicationsequences, wherein the replication sequences mediate replication of thenucleic acid molecules.
 384. The method of claim 383 wherein the nucleicacid molecules are replicated in vitro.
 385. The method of claim 383wherein the nucleic acid molecules are replicated in vivo.
 386. Themethod of claim 385 wherein the nucleic acid molecules are replicated incell culture.
 387. The method of claim 296 wherein each nucleic acidmolecule further comprises integration sequences, wherein theintegration sequences mediate integration of the nucleic acid moleculesinto other nucleic acids.
 388. The method of claim 387 wherein thenucleic acid molecules are integrated into a chromosome.
 389. The methodof claim 388 wherein the nucleic acid molecules are integrated into achromosome at a predetermined location.
 390. The method of claim 296wherein the nucleic acids molecules are produced by replicating nucleicacids in one or more nucleic acid samples.
 391. The method of claim 390wherein the nucleic acids are replicated using pairs of primers, whereineach of the first primers in the primer pairs used to produce thenucleic acid molecules comprises a nucleotide sequence encoding thereporter signal peptide.
 392. The method of claim 391 wherein each firstprimer further comprises expression sequences.
 393. The method of claim392 wherein the nucleotide sequence of each first primer also encodes anepitope tag.
 394. The method of claim 296 wherein each amino acidsegment further comprises an epitope tag.
 395. The method of claim 394wherein the epitope tag of each amino acid segment is different. 396.The method of claim 394 wherein the epitope tag of each amino acidsegment is the same.
 397. The method of claim 394 wherein the epitopetag of at least two amino acid segments are different.
 398. The methodof claim 394 wherein the epitope tag of at least two amino acid segmentsare the same.
 399. The method of claim 394 wherein the amino acidsegments are distinguished or separated from the one or more expressionsamples via the epitope tags.
 400. The method of claim 296 wherein thereporter signal peptide of each amino acid segment is different. 401.The method of claim 296 wherein the reporter signal peptide of eachamino acid segment is the same.
 402. The method of claim 296 wherein thereporter signal peptide of at least two amino acid segments aredifferent.
 403. The method of claim 296 wherein the reporter signalpeptide of at least two amino acid segments are the same.
 404. Themethod of claim 296 wherein the nucleic acid molecules are in cells.405. The method of claim 404 wherein each nucleic acid molecule is in adifferent cell.
 406. The method of claim 404 wherein each nucleic acidmolecule is in the same cell.
 407. The method of claim 406 wherein eachnucleic acid molecule further comprises expression sequences, whereinthe expression sequences are operably linked to the nucleotide segmentsuch that the amino acid segment can be expressed.
 408. The method ofclaim 407 wherein the expression sequences of each nucleic acid moleculeare different.
 409. The method of claim 408 wherein the expressionsequences are similarly regulated.
 410. The method of claim 409 whereina plurality of the expression sequences are expression sequences of, orderived from, genes expressed as part of the same expression cascade.411. The method of claim 404 wherein the nucleic acid molecules areintegrated into a chromosome of the cell.
 412. The method of claim 411wherein the nucleic acid molecules are integrated into the chromosome ata predetermined location.
 413. The method of claim 411 wherein thechromosome is an artificial chromosome.
 414. The method of claim 404wherein the nucleic acid molecules are, or are integrated into, aplasmid.
 415. The method of claim 404 wherein the cells are in celllines.
 416. The method of claim 415 wherein each nucleic acid moleculeis in a different cell line.
 417. The method of claim 415 wherein eachnucleic acid molecule is in the same cell line.
 418. The method of claim404 wherein the expression samples are produced from the cells.
 419. Themethod of claim 418 wherein each expression sample is produced fromcells from a cell sample, wherein each expression sample is producedfrom a different cell sample.
 420. The method of claim 419 wherein eachcell sample is subjected to different conditions.
 421. The method ofclaim 420 wherein each cell sample is brought into contact with adifferent test compound.
 422. The method of claim 420 wherein each cellsample is cultured under different conditions.
 423. The method of claim420 wherein each cell sample is derived from a different organism. 424.The method of claim 420 wherein each cell sample is derived from adifferent tissue.
 425. The method of claim 420 wherein each cell sampleis taken from the same source at different times.
 426. The method ofclaim 418 wherein the expression samples are produced by lysing thecells.
 427. The method of claim 296 wherein the nucleic acid moleculesare in organisms.
 428. The method of claim 427 wherein each nucleic acidmolecule is in a different organism.
 429. The method of claim 427wherein each nucleic acid molecule is in the same organism.
 430. Themethod of claim 429 wherein each nucleic acid molecule further comprisesexpression sequences, wherein the expression sequences are operablylinked to the nucleotide segment such that the amino acid segment can beexpressed.
 431. The method of claim 430 wherein the expression sequencesof each nucleic acid molecule are different.
 432. The method of claim431 wherein the expression sequences are similarly regulated.
 433. Themethod of claim 432 wherein a plurality of the expression sequences areexpression sequences of, or derived from, genes expressed as part of thesame expression cascade.
 434. The method of claim 427 wherein thenucleic acid molecules are integrated into a chromosome of the organism.435. The method of claim 434 wherein the nucleic acid molecules areintegrated into the chromosome at a predetermined location.
 436. Themethod of claim 434 wherein the chromosome is an artificial chromosome.437. The method of claim 427 wherein the nucleic acid molecules are, orare integrated into, a plasmid.
 438. The method of claim 427 whereineach nucleic acid molecule is in a different organism.
 439. The methodof claim 427 wherein each nucleic acid molecule is in the same organism.440. The method of claim 296 wherein the nucleic acid molecules are incells of an organism.
 441. The method of claim 440 wherein the nucleicacid molecules are in substantially all of the cells of the organism.442. The method of claim 440 wherein the nucleic acid molecules are insome of the cells of the organism.
 443. The method of claim 440 whereinthe amino acid segments are expressed in substantially all of the cellsof the organism.
 444. The method of claim 440 wherein the amino acidsegments are expressed in some of the cells of the organism.
 445. Themethod of claim 296 wherein the protein or peptide of interest of eachamino acid segment is different.
 446. The method of claim 296 whereinthe protein or peptide of interest of each amino acid segment is thesame.
 447. The method of claim 296 wherein the protein or peptide ofinterest of at least two amino acid segments are different.
 448. Themethod of claim 296 wherein the protein or peptide of interest of atleast two amino acid segments are the same.
 449. The method of claim 445wherein the proteins or peptides of interest are related.
 450. Themethod of claim 449 wherein the proteins or peptides of interest areproteins produced in the same cascade.
 451. The method of claim 449wherein the proteins or peptides of interest are proteins expressedunder the same conditions.
 452. The method of claim 449 wherein theproteins or peptides of interest are proteins associated with the samedisease.
 453. The method of claim 449 wherein the proteins or peptidesof interest are proteins associated with the same cell type.
 454. Themethod of claim 449 wherein the proteins or peptides of interest areproteins associated with the same tissue type.
 455. The method of claim449 wherein the proteins or peptides of interest are proteins in thesame enzymatic pathway.
 456. The method of claim 296 wherein thenucleotide segment encodes a plurality of amino acid segments eachcomprising a reporter signal peptide and a protein or peptide ofinterest.
 457. The method of claim 456 wherein the protein or peptide ofinterest of at least two of the amino acid segments in one of thenucleotide segments are different.
 458. The method of claim 456 whereinthe protein or peptide of interest of the amino acid segments in one ofthe nucleotide segments are different.
 459. The method of claim 456wherein the protein or peptide of interest of at least two of the aminoacid segments in each of the nucleotide segments are different.
 460. Themethod of claim 456 wherein the protein or peptide of interest of theamino acid segments in each of the nucleotide segments are different.461. The method of claim 456 wherein the set consists of a singlenucleic acid molecule.
 462. The method of claim 296 wherein the setconsists of a single nucleic acid molecule, wherein the nucleic acidmolecule comprises a plurality of nucleotide segments each encoding anamino acid segment.
 463. The method of claim 296 wherein the amino acidsegment comprises a cleavage site near the junction between the reportersignal peptide and the protein or peptide of interest.
 464. The methodof claim 463 wherein the cleavage site is cleaved.
 465. The method ofclaim 464 wherein the reporter signal peptide is distinguished orseparated from the peptide or protein of interest.
 466. The method ofclaim 463 wherein the cleavage site is a trypsin cleavage site.
 467. Themethod of claim 463 wherein the cleavage site is at the junction betweenthe reporter signal peptide and the protein or peptide of interest. 468.The method of claim 296 wherein each amino acid segment furthercomprises a self-cleaving segment.
 469. The method of claim 468 whereinthe self-cleaving segment is between the reporter signal peptide and theprotein or peptide of interest.
 470. The method of claim 469 wherein theself-cleaving segment cleaves the amino acid segment.
 471. The method ofclaim 470 wherein the reporter signal peptide is distinguished orseparated from the peptide or protein of interest.
 472. The method ofclaim 468 wherein the self-cleaving segment is an intein segment. 473.The method of claim 296 wherein a plurality of different alteredreporter signal peptides are detected, wherein detection of each alteredreporter signal peptide indicates expression of the amino acid segmentthat comprises the reporter signal peptide from which that alteredreporter signal peptide is derived.
 474. The method of claim 473 whereindifferent expression samples comprise different nucleic acid molecules,wherein detection of each altered reporter signal peptide indicatesexpression in the expression sample that comprises the nucleic acidmolecule that comprises the nucleotide segment encoding the amino acidsegment that comprises the reporter signal peptide from which thataltered reporter signal peptide is derived.
 475. The method of claim 296wherein there are a plurality of different expression samples, whereineach different expression sample comprises different nucleic acidmolecules, wherein detection of an altered reporter signal peptideindicates expression in the expression sample that comprises the nucleicacid molecule that comprises the nucleotide segment encoding the aminoacid segment that comprises the reporter signal peptide from which thedetected altered reporter signal peptide is derived.
 476. A method ofdetecting expression, the method comprising detecting a target alteredreporter signal peptide derived from one or more expression samples,wherein the one or more expression samples collectively comprise a setof nucleic acid molecules, wherein each nucleic acid molecule comprisesa nucleotide segment encoding an amino acid segment comprising areporter signal peptide and a protein or peptide of interest, whereinthe reporter signal peptides have a common property, wherein the commonproperty allows the reporter signal peptides to be distinguished orseparated from molecules lacking the common property, wherein thereporter signal peptides can be altered, wherein the altered form ofeach reporter signal peptide can be distinguished from the altered formsof the other reporter signal peptides, wherein the target alteredreporter signal peptide is one of the altered reporter signal peptides,wherein detection of the target altered reporter signal peptideindicates expression of the nucleotide segment encoding the amino acidsegment that comprises the reporter signal peptide from which the targetaltered reporter signal peptide is derived.
 477. The method of claim 476further comprising determining the amount of the target altered reportersignal peptide detected, wherein the amount of the target alteredreporter signal peptide indicates the amount present in the one or moreexpression samples of the nucleotide segment that comprises the reportersignal peptide from which the target altered reporter signal peptide isderived.
 478. The method of claim 477 wherein the amount of thenucleotide segment present is proportional to the amount of the targetaltered reporter signal peptide detected.
 479. The method of claim 476further comprising detecting a plurality of the altered reporter signalpeptides, wherein detection of each altered reporter signal peptideindicates expression of the nucleotide segment that comprises thereporter signal peptide from which that altered reporter signal peptideis derived.
 480. The method of claim 479 further comprising determiningthe amount of the altered reporter signal peptides detected, wherein theamount of each altered reporter signal peptide indicates the amountpresent in the one or more expression samples of the nucleotide segmentthat comprises the reporter signal peptide from which that alteredreporter signal peptide is derived.
 481. The method of claim 480 whereinthe amount of the nucleotide segment present is proportional to theamount of the altered reporter signal peptide detected.
 482. A method ofdetecting expression, the method comprising detecting a target alteredamino acid segment derived from one or more expression samples, whereinthe one or more expression samples collectively comprise a set ofnucleic acid molecules, wherein each nucleic acid molecule comprises anucleotide segment encoding an amino acid segment comprising a reportersignal peptide and a protein or peptide of interest, wherein the aminoacid segments have a common property, wherein the common property allowsthe amino acid segments to be distinguished or separated from moleculeslacking the common property, wherein the reporter signal peptides can bealtered, wherein alteration of the reporter signal peptides alters theamino acid segments, wherein the altered form of each amino acid segmentcan be distinguished from the altered forms of the other amino acidsegments, wherein the target altered amino acid segment is one of thealtered amino acid segments, wherein detection of the target alteredamino acid segment indicates expression of the amino acid segment fromwhich the target altered amino acid segment is derived.
 483. A method ofdetecting expression, the method comprising detecting an altered aminoacid subsegment derived from one or more expression samples, wherein theone or more expression samples collectively comprise a set of nucleicacid molecules, wherein each nucleic acid molecule comprises anucleotide segment encoding an amino acid segment comprising a reportersignal peptide and a protein or peptide of interest, wherein the aminoacid segments each comprise an amino acid subsegment, wherein each aminoacid subsegment comprises a portion of the protein or peptide ofinterest and all or a portion of the reporter signal peptide, whereinthe amino acid subsegments have a common property, wherein the commonproperty allows the amino acid subsegments to be distinguished orseparated from molecules lacking the common property, wherein thereporter signal peptides can be altered, wherein alteration of thereporter signal peptides alters the amino acid subsegments, wherein thealtered form of each amino acid subsegment can be distinguished from thealtered forms of the other amino acid subsegments, wherein the targetaltered amino acid subsegment is one of the altered amino acidsubsegments, wherein detection of the target altered amino acidsubsegment indicates expression of the amino acid segment from which thetarget altered amino acid subsegment is derived.
 484. A method ofdetecting cells, the method comprising detecting a target alteredreporter signal peptide derived from one or more cells, wherein the oneor more cells collectively comprise a set of nucleic acid molecules,wherein each nucleic acid molecule comprises a nucleotide segmentencoding an amino acid segment comprising a reporter signal peptide anda protein or peptide of interest, wherein the reporter signal peptideshave a common property, wherein the common property allows the reportersignal peptides to be distinguished or separated from molecules lackingthe common property, wherein the reporter signal peptides can bealtered, wherein the altered form of each reporter signal peptide can bedistinguished from the altered forms of the other reporter signalpeptides, wherein the target altered reporter signal peptide is one ofthe altered reporter signal peptides, wherein detection of the targetaltered reporter signal peptide indicates the presence of the cell fromwhich the target altered reporter signal peptide is derived.
 485. Themethod of claim 484 wherein each cell is engineered to contain at leastone of the nucleic acid molecules, wherein the reporter signal peptideof the amino acid segment encoded by the nucleotide segment of thenucleic acid molecule in each cell is different.
 486. The method ofclaim 485 wherein each cell having a trait of interest comprises thesame reporter signal peptide.
 487. The method of claim 486 wherein thetrait of interest is a heterologous gene.
 488. The method of claim 487wherein the heterologous gene comprises the nucleic acid molecule. 489.The method of claim 486 wherein the heterologous gene encodes the aminoacid segment.
 490. The method of claim 484 wherein a plurality ofdifferent altered reporter signal peptides are detected, whereindetection of each altered reporter signal peptide indicates the presenceof the cell from which that altered reporter signal peptide is derived.491. The method of claim 490 wherein different cells comprise differentnucleic acid molecules, wherein detection of each altered reportersignal peptide indicates the presence of the cell that comprises thenucleic acid molecule that comprises the nucleotide segment encoding theamino acid segment that comprises the reporter signal peptide from whichthat altered reporter signal peptide is derived.
 492. The method ofclaim 484 wherein there are a plurality of different cells, wherein eachdifferent cell comprises different nucleic acid molecules, whereindetection of an altered reporter signal peptide indicates the presenceof the cell that comprises the nucleic acid molecule that comprises thenucleotide segment encoding the amino acid segment that comprises thereporter signal peptide from which the detected altered reporter signalpeptide is derived.
 493. A method of detecting cell samples, the methodcomprising detecting a target altered reporter signal peptide derivedfrom one or more cell samples, wherein the one or more cell samplescollectively comprise a set of nucleic acid molecules, wherein eachnucleic acid molecule comprises a nucleotide segment encoding an aminoacid segment comprising a reporter signal peptide and a protein orpeptide of interest, wherein the reporter signal peptides have a commonproperty, wherein the common property allows the reporter signalpeptides to be distinguished or separated from molecules lacking thecommon property, wherein the reporter signal peptides can be altered,wherein the altered form of each reporter signal peptide can bedistinguished from the altered forms of the other reporter signalpeptides, wherein the target altered reporter signal peptide is one ofthe altered reporter signal peptides, wherein detection of the targetaltered reporter signal peptide indicates the presence of the cellsample from which the target altered reporter signal peptide is derived.494. The method of claim 493 wherein a plurality of different alteredreporter signal peptides are detected, wherein detection of each alteredreporter signal peptide indicates the presence of the cell sample fromwhich that altered reporter signal peptide is derived.
 495. The methodof claim 493 wherein different cell samples comprise different nucleicacid molecules, wherein detection of each altered reporter signalpeptide indicates the presence of the cell sample that comprises thenucleic acid molecule that comprises the nucleotide segment encoding theamino acid segment that comprises the reporter signal peptide from whichthat altered reporter signal peptide is derived.
 496. The method ofclaim 493 wherein there are a plurality of different cell samples,wherein each different cell sample comprises different nucleic acidmolecules, wherein detection of an altered reporter signal peptideindicates the presence of the cell sample that comprises the nucleicacid molecule that comprises the nucleotide segment encoding the aminoacid segment that comprises the reporter signal peptide from which thedetected altered reporter signal peptide is derived.
 497. A method ofdetecting cells, the method comprising detecting a target alteredreporter signal peptide derived from one or more cells, wherein the oneor more cells collectively comprise a set of nucleic acid molecules,wherein each nucleic acid molecule comprises a nucleotide segmentencoding an amino acid segment comprising a reporter signal peptide anda protein or peptide of interest, wherein the reporter signal peptideshave a common property, wherein the common property allows the reportersignal peptides to be distinguished or separated from molecules lackingthe common property, wherein the reporter signal peptides can bealtered, wherein the altered form of each reporter signal peptide can bedistinguished from the altered forms of the other reporter signalpeptides, wherein the target altered reporter signal peptide is one ofthe altered reporter signal peptides, wherein detection of the targetaltered reporter signal peptide indicates the presence of the cell fromwhich the target altered reporter signal peptide is derived.
 498. Amethod of detecting cells, the method comprising detecting a targetaltered amino acid segment derived from one or more cells, wherein theone or more cells collectively comprise a set of nucleic acid molecules,wherein each nucleic acid molecule comprises a nucleotide segmentencoding an amino acid segment comprising a reporter signal peptide anda protein or peptide of interest, wherein the amino acid segments have acommon property, wherein the common property allows the amino acidsegments to be distinguished or separated from molecules lacking thecommon property, wherein the reporter signal peptides can be altered,wherein alteration of the reporter signal peptides alters the amino acidsegments, wherein the altered form of each amino acid segment can bedistinguished from the altered forms of the other amino acid segments,wherein the target altered amino acid segment is one of the alteredamino acid segments, wherein detection of the target altered amino acidsegment indicates the presence of the cell from which the target alteredamino acid segment is derived.
 499. A method of detecting cells, themethod comprising detecting an altered amino acid subsegment derivedfrom one or more cells, wherein the one or more cells collectivelycomprise a set of nucleic acid molecules, wherein each nucleic acidmolecule comprises a nucleotide segment encoding an amino acid segmentcomprising a reporter signal peptide and a protein or peptide ofinterest, wherein the amino acid segments each comprise an amino acidsubsegment, wherein each amino acid subsegment comprises a portion ofthe protein or peptide of interest and all or a portion of the reportersignal peptide, wherein the amino acid subsegments have a commonproperty, wherein the common property allows the amino acid subsegmentsto be distinguished or separated from molecules lacking the commonproperty, wherein the reporter signal peptides can be altered, whereinalteration of the reporter signal peptides alters the amino acidsubsegments, wherein the altered form of each amino acid subsegment canbe distinguished from the altered forms of the other amino acidsubsegments, wherein the target altered amino acid subsegment is one ofthe altered amino acid subsegments, wherein detection of the targetaltered amino acid subsegment indicates the presence of the cell fromwhich the target altered amino acid subsegment is derived.
 500. A methodof detecting organisms, the method comprising detecting a target alteredreporter signal peptide derived from one or more organisms, wherein theone or more organisms collectively comprise a set of nucleic acidmolecules, wherein each nucleic acid molecule comprises a nucleotidesegment encoding an amino acid segment comprising a reporter signalpeptide and a protein or peptide of interest, wherein the reportersignal peptides have a common property, wherein the common propertyallows the reporter signal peptides to be distinguished or separatedfrom molecules lacking the common property, wherein the reporter signalpeptides can be altered, wherein the altered form of each reportersignal peptide can be distinguished from the altered forms of the otherreporter signal peptides, wherein the target altered reporter signalpeptide is one of the altered reporter signal peptides, whereindetection of the target altered reporter signal peptide indicates thepresence of the organism from which the target altered reporter signalpeptide is derived.
 501. The method of claim 500 wherein each organismis engineered to contain at least one of the nucleic acid molecules,wherein the reporter signal peptide of the amino acid segment encoded bythe nucleotide segment of the nucleic acid molecule in each organism isdifferent.
 502. The method of claim 501 wherein each organism having atrait of interest comprises the same reporter signal peptide.
 503. Themethod of claim 502 wherein the trait of interest is a transgene. 504.The method of claim 503 wherein the transgene gene comprises the nucleicacid molecule.
 505. The method of claim 502 wherein the transgene geneencodes the amino acid segment.
 506. The method of claim 500 wherein aplurality of different altered reporter signal peptides are detected,wherein detection of each altered reporter signal peptide indicates thepresence of the organism from which that altered reporter signal peptideis derived.
 507. The method of claim 506 wherein different organismscomprise different nucleic acid molecules, wherein detection of eachaltered reporter signal peptide indicates the presence of the organismthat comprises the nucleic acid molecule that comprises the nucleotidesegment encoding the amino acid segment that comprises the reportersignal peptide from which that altered reporter signal peptide isderived.
 508. The method of claim 500 wherein there are a plurality ofdifferent organisms, wherein each different organism comprises differentnucleic acid molecules, wherein detection of an altered reporter signalpeptide indicates the presence of the organism that comprises thenucleic acid molecule that comprises the nucleotide segment encoding theamino acid segment that comprises the reporter signal peptide from whichthe detected altered reporter signal peptide is derived.
 509. A methodof detecting organisms, the method comprising detecting a target alteredreporter signal peptide derived from one or more organisms, wherein theone or more organisms collectively comprise a set of nucleic acidmolecules, wherein each nucleic acid molecule comprises a nucleotidesegment encoding an amino acid segment comprising a reporter signalpeptide and a protein or peptide of interest, wherein the reportersignal peptides have a common property, wherein the common propertyallows the reporter signal peptides to be distinguished or separatedfrom molecules lacking the common property, wherein the reporter signalpeptides can be altered, wherein the altered form of each reportersignal peptide can be distinguished from the altered forms of the otherreporter signal peptides, wherein the target altered reporter signalpeptide is one of the altered reporter signal peptides, whereindetection of the target altered reporter signal peptide indicates thepresence of the organism from which the target altered reporter signalpeptide is derived.
 510. A method of detecting organisms, the methodcomprising detecting a target altered amino acid segment derived fromone or more organisms, wherein the one or more organisms collectivelycomprise a set of nucleic acid molecules, wherein each nucleic acidmolecule comprises a nucleotide segment encoding an amino acid segmentcomprising a reporter signal peptide and a protein or peptide ofinterest, wherein the amino acid segments have a common property,wherein the common property allows the amino acid segments to bedistinguished or separated from molecules lacking the common property,wherein the reporter signal peptides can be altered, wherein alterationof the reporter signal peptides alters the amino acid segments, whereinthe altered form of each amino acid segment can be distinguished fromthe altered forms of the other amino acid segments, wherein the targetaltered amino acid segment is one of the altered amino acid segments,wherein detection of the target altered amino acid segment indicates thepresence of the organism from which the target altered amino acidsegment is derived.
 511. A method of detecting organisms, the methodcomprising detecting an altered amino acid subsegment derived from oneor more organisms, wherein the one or more organisms collectivelycomprise a set of nucleic acid molecules, wherein each nucleic acidmolecule comprises a nucleotide segment encoding an amino acid segmentcomprising a reporter signal peptide and a protein or peptide ofinterest, wherein the amino acid segments each comprise an amino acidsubsegment, wherein each amino acid subsegment comprises a portion ofthe protein or peptide of interest and all or a portion of the reportersignal peptide, wherein the amino acid subsegments have a commonproperty, wherein the common property allows the amino acid subsegmentsto be distinguished or separated from molecules lacking the commonproperty, wherein the reporter signal peptides can be altered, whereinalteration of the reporter signal peptides alters the amino acidsubsegments, wherein the altered form of each amino acid subsegment canbe distinguished from the altered forms of the other amino acidsubsegments, wherein the target altered amino acid subsegment is one ofthe altered amino acid subsegments, wherein detection of the targetaltered amino acid subsegment indicates the presence of the organismfrom which the target altered amino acid subsegment is derived.
 512. Amethod comprising (a) associating one of a plurality of reporter signalswith one or more analytes in each of a plurality of samples, whereineach reporter signal has a common property, wherein the common propertyallows each reporter signal to be separated from molecules lacking thecommon property, (b) separating the analytes contained in each sample,(c) altering the reporter signals, and (d) detecting the altered formsthe reporter signals.
 513. The method of claim 512 further comprising,following step (a) and prior to step (b), combining two or more of thesamples.
 514. The method of claim 512 wherein analytes in each sampleare associated with only one reporter signal, wherein the reportersignal associated with analytes in each sample is different.
 515. Themethod of claim 512 wherein the analytes are separated by contact with acapture array.
 516. A method comprising (a) associating one of aplurality of coding tags with one or more analytes in at least onesample, (b) separating the analytes contained in each sample; (c)associating the coding tags with one or more reporter molecules, whereineach reporter molecule comprises a reporter signal and a decoding tag,wherein each reporter signal has a common property, wherein the commonproperty allows each reporter signal to be separated from moleculeslacking the common property, wherein each decoding tag is specific forone or more of the coding tags, (d) altering the reporter signals, and(e) detecting the altered forms the reporter signals.
 517. The method ofclaim 516 wherein the coding tags are oligonucleotides, wherein thedecoding tags are peptide nucleic acids, and wherein the reportersignals are reporter signal peptides.
 518. A method comprising (a)associating one of a plurality of coding tags with one or more analytesin at least one sample, (b) separating the analytes contained in eachsample; (c) associating the coding tags with one or more reportersignals, wherein each reporter signal has a common property, wherein thecommon property allows each reporter signal to be separated frommolecules lacking the common property, wherein each reporter signal isspecific for one or more of the coding tags, (d) altering the reportersignals, and (e) detecting the altered forms the reporter signals. 519.The method of claim 518 wherein the coding tags are oligonucleotides,and wherein the reporter signals are peptide nucleic acids.
 520. Amethod comprising (a) associating one of a plurality of reporter signalswith one or more analytes in each of a plurality of samples to formreporter signal/analyte conjugates, wherein each reporter signal/analyteconjugate has a common property, wherein the common property allows eachreporter signal/analyte conjugate to be separated from molecules lackingthe common property, (b) separating the analytes contained in eachsample, wherein separation is not based on the common property of thereporter signal/analyte conjugates, (c) altering the reporter signalsthereby altering the reporter signal/analyte conjugates, and (d)detecting the altered forms the reporter signal/analyte conjugates. 521.A method comprising (a) associating one of a plurality of coding tagswith one or more analytes in at least one sample, (b) separating theanalytes contained in each sample; (c) associating the coding tags withone or more reporter molecules, wherein each reporter molecule comprisesa reporter signal and a decoding tag, wherein each reporter molecule hasa common property, wherein the common property allows each reportermolecule to be separated from molecules lacking the common property,wherein each decoding tag is specific for one or more of the codingtags, (d) altering the reporter signals thereby altering the reportermolecules, and (e) detecting the altered forms the reporter molecules.